Neurosteroid analysis by gas chromatography–atmospheric pressure photoionization–tandem mass spectrometry

A new and simple APPI interface employing commercially available hardware is used to combine GC to MS. The feasibility of the method is demonstrated in the analysis of urine samples for neurosteroids as their trimethylsilyl (TMS) derivatives. The effect of different dopants (chlorobenzene, toluene, anisole) on the ionization of the TMS derivatives was investigated. With chlorobenzene, the TMS derivatives produced intense molecular ions with minimal fragmentation, and chlorobenzene was selected as best dopant. Protonated molecules in addition to intense molecular ions were produced with toluene and anisole. The performance of the method was verified in the analysis of human urine samples. Chromatographic performance was good with peak half-widths of 3.6–4.3 s, linearity (r² > 0.990) was acceptable, limits of detection (LODs) were in the range of 0.01–10 ng mL⁻¹, and repeatability was good with relative standard deviations (rsd%) below 22%. The results show that the method is well suited for the determination of neurosteroids in biological samples.     

Application of pentafluorophenyl hydrazine derivatives to the analysis of nabumetone and testosterone in human plasma by liquid chromatography–atmospheric pressure chemical ionization–tandem mass spectrometry

Two carbonyl compounds, nabumetone and testosterone, were derivatized with pentafluorophenyl hydrazine (PFPH) and analyzed by atmospheric-pressure chemical-ionization mass spectrometry. The PFPH derivatives underwent dissociative electron capture in negative-ion APCI (ECAPCI) and gave intense [M–20]^– ions in the mass spectra. In positive-ion APCI, the PFPH derivatives underwent efficient protonation and gave intense [M+H]⁺ ions in the mass spectra. In CID, the major product ions of the [M–20]^– ions in ECAPCI corresponded to the partial moiety of PFPH. In contrast, the major product ions of [M+H]⁺ corresponded to the partial moiety of the analyte. By using selected reaction monitoring (SRM) detection, low pg of nabumetone (1 pg) and testosterone (7 pg) could be detected in both ECAPCI and positive-ion APCI. In comparison with the detection limits (SRM) of the underivatized analytes, use of the PFPH derivatives resulted in 2500-fold and 35-fold sensitivity enhancements for nabumetone and testosterone, respectively. The PFPH derivatives were applied to the analysis of nabumetone and testosterone in human plasma by both ECAPCI and positive-ion APCI and were found to enable detection of 0.1 ng mL^–1 nabumetone in spiked plasma. For testosterone, endogenous testosterone in female plasma was detected in both ECAPCI and positive-ion APCI.     

Pharmaceutical trace analysis in aqueous environmental matrices by liquid chromatography–ion trap tandem mass spectrometry

An analytical method based on solid-phase extraction followed by liquid chromatography tandem mass spectrometry with an ion trap analyser was developed and validated for the quantification of a series of pharmaceutical compounds with distinct physical–chemical characteristics in estuarine water samples. Method detection limits were between 0.03 and 16.4 ng/L. The sensitivity and the accuracy obtained associated with the inherent confirmatory potential of ion trap tandem mass spectrometry (IT-MS/MS) validates its success as an environmental analysis tool. Two MS/MS transitions were used to confirm compound identity. Almost all pharmaceuticals were detected at ng/L level in at least one sampling site of the Douro River estuary, Portugal.     

Determination of biocides in different environmental matrices by use of ultra-high-performance liquid chromatography–tandem mass spectrometry

A sensitive and robust method using solid-phase extraction and ultrasonic extraction for preconcentration followed by ultra-high-performance liquid chromatography–tandem mass spectrometry (UHPLC–MS–MS) has been developed for determination of 19 biocides: eight azole fungicides (climbazole, clotrimazole, ketoconazole, miconazole, fluconazole, itraconazole, thiabendazole, and carbendazim), two insect repellents (N,N-diethyl-3-methylbenzamide (DEET), and icaridin (also known as picaridin)), three isothiazolinone antifouling agents (1,2-benzisothiazolinone (BIT), 2-n-octyl-4-isothiazolinone (OIT), and 4,5-dichloro-2-n-octyl-isothiazolinone (DCOIT)), four paraben preservatives (methylparaben, ethylparaben, propylparaben, and butylparaben), and two disinfectants (triclosan and triclocarban) in surface water, wastewater, sediment, sludge, and soil. Recovery of the target compounds from surface water, influent, effluent, sediment, sludge, and soil was mostly in the range 70–120?%, with corresponding method quantification limits ranging from 0.01 to 0.31?ng?L^?1, 0.07 to 7.48?ng?L^?1, 0.01 to 3.90?ng?L^?1, 0.01 to 0.45?ng?g^?1, 0.01 to 6.37?ng?g^?1, and 0.01 to 0.73?ng?g^?1, respectively. Carbendazim, climbazole, clotrimazole, methylparaben, miconazole, triclocarban, and triclosan were detected at low ng?L^?1 (or ng?g^?1) levels in surface water, sediment, and sludge-amended soil. Fifteen target compounds were found in influent samples, at concentrations ranging between 0.4 (thiabendazole) and 372?ng?L^?1 (methylparaben). Fifteen target compounds were found in effluent samples, at concentrations ranging between 0.4 (thiabendazole) and 114?ng?L^?1 (carbendazim). Ten target compounds were found in dewatered sludge samples, at concentrations ranging between 1.1 (DEET) and 887?ng?g^?1 (triclocarban).     

Comparison of supercritical fluid chromatography–tandem mass spectrometry and liquid chromatography–tandem mass spectrometry for the stereoselective analysis of chlorfenvinphos and dimethylvinphos in tobacco

This study used reversed-phase liquid chromatography–tandem mass spectrometry and supercritical fluid chromatography–tandem mass spectrometry for determination of the stereoisomers of chlorfenvinphos and dimethylvinphos in tobacco. Tobacco samples were extracted and purified with a modified quick, easy, cheap, effective, rugged, and safe technique using spherical carbon. The performance of both methodologies was comprehensively compared in terms of methods validation parameters (separation efficiency, linearity, selectivity, recovery, repeatability, sensitivity, matrix effect, etc.). Under optimized conditions, the calibration curves of the stereoisomers of chlorfenvinphos and dimethylvinphos in the range of 10–500 ng/mL showed excellent linearity with R² ≥ 0.997 in both methods. The adequate recoveries of analytes from three different spiked tobaccos were obtained using reversed-phase liquid chromatography–tandem mass spectrometry (86.1–95.7%) as well as supercritical fluid chromatography–tandem mass spectrometry (86.5–94.0%). The relative standard deviations for spiked samples were all below 7.0%. Compared with supercritical fluid chromatography–tandem mass spectrometry, lower matrix effects and LODs can be obtained in reversed-phase liquid chromatography–tandem mass spectrometry.     

Determination of pharmaceuticals from different therapeutic classes in wastewaters by liquid chromatography–electrospray ionization–tandem mass spectrometry

A sensitive analytical method has been developed and validated for simultaneous determination of pharmaceuticals from different therapeutic classes, i.e. five sulfonamide (SA) and trimethoprim antimicrobials and the anti-inflammatory drug diclofenac, in effluent wastewaters at trace levels. Effluent samples from treatment of wastewater were enriched by solid-phase extraction (SPE) using the Waters Oasis HLB cartridge. The analytes were identified and quantified by reversed-phase liquid chromatography-tandem mass spectrometry operated in the selected reaction monitoring (SRM) mode, using positive electrospray ionization. The pharmaceuticals were, consequently, quantified both by use of isotopically labelled internal standards and by standard addition methods to address the issue of matrix effects related to signal suppression by co-eluting compounds. Average recoveries from fortified samples were usually >70%, with relative standard deviations below 20%. Method detection limits in wastewater matrices were between 7.0 and 10 ng L(-1). Identification points (IPs) were used for unequivocal identification of target analytes in real samples. Diclofenac, trimethoprim, and sulfamethoxazole were mainly detected, in the concentration range 10 to 400 ng L(-1), in effluent samples collected from four different sewage-treatment plants in Greece.     

Quantification of clomiphene metabolite isomers in human plasma by rapid-resolution liquid chromatography–electrospray ionization–tandem mass spectrometry

Since the 1960s, clomiphene citrate is used for ovulation induction. Since nonresponse to clomiphene therapy is still not well understood, interindividual variability of clomiphene metabolism has been considered to be a plausible explanation. Therefore, a comprehensive, rapid, sensitive, and specific analytical method for the quantification of (E)- and (Z)-isomers of clomiphene and their putative N-desethyl, N,N-didesethyl, 4-hydroxy, and 4-hydroxy-N-desethyl metabolites, and the N-oxides in human plasma has been newly developed, using HPLC-tandem mass spectrometry and stable isotope-labeled internal standards. All standards other than the parent drug were synthesized in our laboratory. Following protein precipitation analytes were separated on a ZORBAX Eclipse plus C18 1.8 μm column with a gradient of 0.1% formic acid in water and 0.1% formic acid in acetonitrile and detected on a triple quadrupole mass spectrometer with positive electrospray ionization in the multiple reaction monitoring mode. Lower limit of quantification for metabolites ranged from 0.06 ng/mL for clomiphene-N-oxides to 0.3 ng/mL for (E)-N-desethylclomiphene. The assay was validated according to FDA guidelines. Plasma levels of clomiphene and its metabolites were measured in two women after single-dose treatment with clomiphene.     

Development and validation of a multi-residue method for the determination of pesticides in processed fruits and vegetables using liquid chromatography–electrospray ionization tandem mass spectrometry

A sensitive multi-residue analytical method, utilizing ethyl acetate extraction and liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS-MS), has been developed and validated for simultaneous determination of 28 pesticides of different chemical classes (polar organophosphates, carbamates, strobilurines, neonicotinoids, amides, pyrimidines, benzimidazoles, imidazoles and triazoles), and their transformation products, in processed fruit and vegetables. Two precursor-product ion transitions were monitored for each pesticide in selected reaction monitoring (SRM) mode. Linearity (r (2) > or = 0.99) was good over the concentration range 0.5 to 100 microg L(-1) for all the pesticides, and instrumental detection limits ranged from 0.1 to 1 microg L(-1). Mean recovery for fruit and vegetables spiked at 0.010 mg kg(-1) ranged from 65 to 94.4%, and relative standard deviations ranged from 9.0 to 20.0%. When the amount spiked was 0.050 mg kg(-1) recoveries ranged from 72.5 to 90% and relative standard deviations were from 6.1 to 19.0%. Method detection limits were from 0.002 to 0.007 mg kg(-1) for the different food matrices studied. The method was used to monitor pesticide residues in a wide variety of fruits and vegetables.     

Enantiomeric determination of azole antifungals in wastewater and sludge by liquid chromatography–tandem mass spectrometry

A sensitive and reliable liquid chromatographic-tandem mass spectrometric method for enantiomeric determination of five chiral azole antifungals (econazole, ketoconazole, miconazole, tebuconazole, and propiconazole) in wastewater and sludge has been established and validated. An isotope-labeled internal standard was used for quantification. Recovery of the individual enantiomers was usually in the range of 77-102 % for wastewater and 71-95 % for sludge, with relative standard deviations within 20 %. No significant difference (p>0.05) was observed between recovery of pairs of enantiomers of the chiral azole antifungals except for those of tebuconazole. Method quantification limits for individual enantiomers were 0.3-10 ng L(-1) and 3-29 ng g(-1) dry weight for wastewater and sludge, respectively. The method was used to investigate the enantiomeric composition of the azole pharmaceuticals in wastewater and sludge samples from a sewage treatment plant in China. Enantiomers of miconazole, ketoconazole, and econazole were widely detected. The results showed that the azole antifungals in wastewater and sludge were generally racemic or marginally non-racemic. The method is a useful tool for investigation of the enantiomeric occurrence, behavior, and fate of the chiral azole antifungals in the environment.     

Resin and fatty-acid analysis by solid-phase extraction coupled to atmospheric pressure chemical ionization–mass spectrometry

Abstract

DNA biosensors are realised immobilising a DNA structure on a suitable transducer to obtain selective information. In this paper we show how the determination of low-molecular weight compounds with affinity for DNA was measured by their effect on the oxidation signal of the guanine peak of calf thymus DNA immobilised on the electrode sensor and investigated by chronopotentiometric analysis. The DNA biosensor is able to detect known intercalating and groove binding compounds. Applicability to river water samples was demostrated.

Moreover, a piezoelectric sensor coupled to a short oligonucleotide can be used as detector of the hybridisation reaction. We show as a model the detection of a specific mutation in apolipoprotein E (ApoE) gene.

Biotinylated 23-mer probes were immobilised on the streptavidin coated gold surface of a quartz crystal; the protein was covalently bound to the thiol/dextran modified gold surface. The device was able to distinguish different synthetic oligonucleotides. The hybridisation reaction was also performed using real samples of DNA extracted from human blood and amplified by Polymerase chain reaction PCR. The extension of such procedure to samples of environmental interest is discussed.     

Trace analysis of environmental matrices by large-volume injection and liquid chromatography–mass spectrometry

The time-honored convention of concentrating aqueous samples by solid-phase extraction (SPE) is being challenged by the increasingly widespread use of large-volume injection (LVI) liquid chromatography–mass spectrometry (LC–MS) for the determination of traces of polar organic contaminants in environmental samples. Although different LVI approaches have been proposed over the last 40 years, the simplest and most popular way of performing LVI is known as single-column LVI (SC-LVI), in which a large-volume of an aqueous sample is directly injected into an analytical column. For the purposes of this critical review, LVI is defined as an injected sample volume that is ≥10% of the void volume of the analytical column. Compared with other techniques, SC-LVI is easier to set up, because it requires only small hardware modifications to existing autosamplers and, thus, it will be the main focus of this review. Although not new, SC-LVI is gaining acceptance and the approach is emerging as a technique that will render SPE nearly obsolete for many environmental applications. In this review, we discuss: the history and development of various forms of LVI; the critical factors that must be considered when creating and optimizing SC-LVI methods; and typical applications that demonstrate the range of environmental matrices to which LVI is applicable, for example drinking water, groundwater, and surface water including seawater and wastewater. Furthermore, we indicate direction and areas that must be addressed to fully delineate the limits of SC-LVI.     

Analysis of aldehydes in beer by gas-diffusion microextraction: Characterization by high-performance liquid chromatography–diode-array detection–atmospheric pressure chemical ionization–mass spectrometry

In this work, a recently developed extraction technique for sample preparation aiming the analysis of volatile and semi-volatile compounds named gas-diffusion microextraction (GDME) is applied in the chromatographic analysis of aldehydes in beer. Aldehydes—namely acetaldehyde (AA), methylpropanal (MA) and furfural (FA)—were simultaneously extracted and derivatized with 2,4-dinitrophenylhydrazine (DNPH), then the derivatives were separated and analyzed by high-performance liquid chromatography with spectrophotometric detection (HPLC–UV). The identity of the eluted compounds was confirmed by high-performance liquid chromatography–atmospheric pressure chemical ionization–mass-spectrometry detection in the negative ion mode (HPLC–APCI–MS). The developed methodology showed good repeatability (ca. 5%) and linearity as well as good limits of detection (AA–12.3, FA–1.5 and MA 5.4 μg L⁻¹) and quantification (AA–41, FA–4.9 and MA 18 μg L⁻¹); it also appears to be competitive in terms of speed and cost of analysis.     

Identification and quantification of salinomycin in intoxicated human plasma by liquid chromatography–electrospray tandem mass spectrometry

Salinomycin is a polyether ionophore antibiotic that is widely used in poultry and livestock. Exposure of humans to salinomycin via inhalation or ingestion can cause severe toxicity. The aim of the present work was to develop a simple and sensitive liquid chromatography–tandem mass spectrometry (LC-MS/MS) method for the rapid identification and quantification of salinomycin in human plasma. After removing protein using methanol, plasma samples were eluted from a Waters Xterra ^® MS C18 column with an isocratic mobile phase. Detection and quantification of the drug were performed with a triple-quadruple mass spectrometer by monitoring for two specific transitions in the electrospray, positive-ion, multiple-reaction monitoring mode. Assay validation showed good linearity (r ²?=?0.998). The detection and quantification limits of the method were 0.6 and 16 pg/mL, respectively. The inter- and intraday coefficients of variation for the assay were both <15%. Twelve authentic plasma samples from intoxicated patients were analyzed using this method. Salinomycin was detected in six samples, at concentrations of between 0.6 and 46.5 pg/mL. The described assay method allows the sensitive and rapid identification and quantification of salinomycin in human plasma, and thus provides a valuable tool for the specific diagnosis of salinomycin intoxication in clinical and emergency rescue practice.     

Residue analysis of glucocorticoids in bovine milk by liquid chromatography–tandem mass spectrometry

A sensitive liquid chromatography–tandem mass spectrometry (LC-MS/MS) method for the simultaneous determination of 13 steroidal anti-inflammatory drugs in bovine milk is presented. Due to their weakly acid nature, analytes were separated by ion suppression reversed phase chromatography and detected in positive-ion mode by a high flow electrospray source. Dexamethasone-d4 was used as internal standard. The sample preparation was simple and reliable; it included acidic deproteinization of milk followed by sample enrichment and clean-up, utilizing a C18 solid phase extraction cartridge. Recoveries exceeded 70% with an intra-day precision not larger than 12%. The efficiency of the sample clean-up and internal standardization rendered negligible the matrix effect, estimated by comparing standard and matrix-matched calibration curves. A small-scale reconnaissance was carried out on several raw and whole fresh milk samples. A large number of analyzed samples showed a chromatographic peak, in the retention time window of cortisol, at levels included between its decision limit (CCα) and detection capability (CCβ). As a result of a heat-induced transformation, an isomeric product of triamcinolone was observed during the extract evaporation. Since this rearrangement might occur during the milk pasteurization process, LC-MS/MS and ¹H-NMR investigations were performed out to conclusively differentiate the two isomers. One- and two-dimensional proton NMR spectra were able to identify the transformation product as 9a-fluoro-11b,16a-trihydroxy-17b-hydroxymethyl-D-homoandrosta-1,4-diene-3,17a-dione.     

Simultaneous determination by ultra-performance liquid chromatography–atmospheric pressure chemical ionization time-of-flight mass spectrometry of nitrated and oxygenated PAHs found in air and soot particles

An ultra-performance liquid chromatographic-atmospheric pressure chemical ionization time-of-flight mass spectrometric (UPLC-APCIToFMS) method for rapid analysis of twelve nitrated polycyclic aromatic hydrocarbons (NPAHs) and nine oxygenated polycyclic aromatic hydrocarbons (OPAHs) in particle samples has been developed. The extraction step using pressurized liquid extraction was optimized by experimental design methods and the concentrated extracts were analyzed without further clean-up. Matrix effects resulting in suppression or enhancement of the response during the ionization step were not observed. The suitability of the developed method is demonstrated by analysis of six different particle samples including standard reference materials, atmospheric particles collected by a high-volume sampler at an urban background site, and a soot sample from a burner. Results from these measurements showed clear differences between the different kinds of samples. Concentrations from reference materials are in good agreement with those from previous studies. Additionally a clear seasonal trend could be observed in atmospheric NPAH and OPAH concentrations found in real samples, with higher concentrations in winter.     

Liquid chromatography–atmospheric pressure photoionization tandem mass spectrometry for analysis of 36 halogenated flame retardants in fish

A comprehensive, sensitive and high-throughput liquid chromatography–atmospheric pressure photoionization tandem mass spectrometry (LC–APPI-MS/MS) method has been developed for analysis of 36 halogenated flame retardants (HFRs). Under the optimized LC conditions, all of the HFRs eluted from the LC column within 14 min, while maintaining good chromatographic separation for the isomers. Introduction of the pre-heated dopant to the APPI source decreased the background noise fivefold, which enhanced sensitivity. An empirical equation was proposed to describe the relation between the ion intensity and dopant flow. The excellent on-column instrument detection limits averaged 4.7 pg, which was similar to the sensitivity offered by gas chromatography–high-resolution mass spectrometry (GC–HRMS). This method was used to analyze a series of fish samples. Good agreement was found between the results for PBDEs from LC–APPI-MS/MS and GC–HRMS.     

Simultaneous determination of benzotriazole and benzothiazole derivatives in aqueous matrices by mixed-mode solid-phase extraction followed by liquid chromatography–tandem mass spectrometry

An improved selectivity method for the simultaneous determination of four benzotriazoles (benzotriazole, 4-methylbenzotriazole, 5-methylbenzotriazole, and 5,6-dimethyl-1H-benzotriazole) and six benzothiazoles (benzothiazole, 2-hydroxybenzothiazole, 2-benzothiazolamine, mercaptobenzothiazole, 2-methylbenzothiazole, and 2-methylthiobenzothiazole) in aqueous matrices has been developed. Under optimal conditions, analytes are concentrated using a MAX solid-phase extraction (SPE) cartridge, based on divinylbenzene-N-vinylpyrrolidone functionalized with quaternary amine groups, which allows reversed-phase interactions in combination with ionic exchange. Selected compounds are recovered with methanol–acetone 7:3 (v/v) whereas acidic interferences remained attached to the sorbent, and as determined by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), LOQs for surface, urban and industrial wastewater are in the range of 0.002–0.29 ng/mL. Figures of merit of the method revealed good precision (RSD% <12%), linearity (R ² > 0.99) and accuracy (%R = 80–100%) for surface waters and effluents allowing direct external standard quantification. For more complex samples, such as urban and industrial raw wastewater, either the standard addition method or pseudo-external standard calibration using matrix matched standards are recommended. Analysis of different real samples, surface, urban wastewater and, for the first time, metal industry wastewater, reflected concentrations up to 310 ng/mL. The methylbenzotriazole isomers ratio was also determined.     

Multiclass method for antimicrobial analysis in animal feeds by liquid chromatography–tandem mass spectrometry

A rapid multiclass method that covers 50 antimicrobials from 13 different families in animal feeds was developed. Samples were extracted using a mixture of methanol, acetonitrile and a McIlvaine buffer combined with sonication. Feed extracts were simply diluted prior to injection, since the clean-up strategies that were tested, based on either solid-phase extraction or dispersive solid-phase extraction, were ineffective at minimizing matrix-related signal suppression/enhancement. Analysis was carried out by liquid chromatography coupled to tandem mass spectrometry using an electrospray ionization source operating in positive and negative modes. For the quantification, matrix-fortified standard calibration curves were used to compensate for matrix effects and losses in sample preparation. The method was validated in-house in pig, poultry and cattle feed matrices and showed satisfactory performance characteristics. Thus, the proposed approach was suitable for application in a routine high-throughput laboratory for the official control of feeds.

Quantitative determination of capsaicinoids by liquid chromatography–electrospray mass spectrometry

Eight naturally occurring capsaicinoids have been determined in Capsicum by use of high-purity standards, with norcapsaicin as an internal standard. The solid standards were rigorously checked for purity. The sensitivity of electrospray ionization (ESI), atmospheric-pressure chemical ionization (APCI), and coordination ion-spray (CIS; with silver) toward the capsaicinoids were measured and compared. The highest sensitivity was found for positive-ion ESI. Method validation of the liquid chromatography–ESI-mass spectrometry (LC–ESI-MS) determination is reported, including tests for repeatability (4%), detection limit (5 pg injected), linear range (20–6 ng injected), quantitation (excellent linearity; <2% relative standard deviation), and recovery (99–103%). The major and minor capsaicinoids in a commercial plant extract and in chili pepper fruits were quantified.Electronic Supplementary Material Supplementary material is available for this article at