Assessment of the occurrence and distribution of pharmaceuticals in a Mediterranean wetland (L'Albufera, Valencia, Spain) by LC-MS/MS

The distribution of 17 pharmaceuticals between water and the solid phase (sediments and soils) was studied by utilizing solid-phase extraction (SPE) and liquid chromatography?Ctandem mass spectrometry (LC-MS/MS). Two extraction procedures for soils and sediments, prior to the SPE, one based on pressurized liquid extraction (PLE) with hot water and the other on methanol/water ultrasonic extraction, were compared. Absolute recoveries were 71.2?C99.3% [relative standard deviation (RSD) <21.4%)] for water, and the method detection limits (MDLs) ranged from 0.3 to 10?ng?L^?1. Recoveries were 35.4?C105.3% (RSDs <19.1%) and 42.1?C97.8% (RSDs <14%) for soil and sediment samples, respectively, using PLE and 20.2?C86.5% (RSDs <25.1%) and 30.3?C97.4% (RSDs <19.1%) using ultrasonic extraction. Fifteen of the 17 pharmaceuticals were present in the L??Albufera water at concentrations up to 17???g?L^?1. Oxytetracycline and tetracycline were not detected. In sediments, only tetracycline, norfloxacin and diclofenac were not found. The other studied pharmaceuticals were present in the range from less than the method quantification limit (MQL) to 35.83?ng?g^?1. Among the 17 target compounds, ofloxacin, ciprofloxacin, norfloxacin, trimethoprim, clofibric acid and diclofenac were not detected in soil samples. The average concentrations ranged from less than the MQL for ibuprofen to 34.91?ng?g^?1 for tetracycline. These results indicate that pharmaceuticals could survive the wastewater treatment processes, which could lead to their dissemination in water environments.     

Simultaneous multi-residue pesticide analysis in soil samples with ultra-high-performance liquid chromatography–tandem mass spectrometry using QuEChERS and pressurised liquid extraction methods

To assess soil contamination, it is important to be able to measure different classes of pesticides simultaneously. For this reason we developed a sensitive ultra-high-performance liquid chromatography–tandem mass spectrometry method for the simultaneous analysis of 25 pesticides in soil samples. Multi-class pesticides (triazines, phenylureas, phenoxy acid pesticides etc.) were analysed using a single mass spectrometry method with a fast polarity switching option, allowing the analysis of 19 compounds in the positive ionisation mode and six compounds in the negative ionisation mode. Extraction of pesticides from soil samples was performed employing a pressurised liquid extraction (PLE) and a quick, easy, cheap, effective, rugged and safe (QuEChERS) procedure, recently developed for the extraction of multi-residue pesticides from food matrices. The extraction efficiency, performance and recoveries of these two procedures were evaluated and compared. In addition, we studied the effect of matrix on signal suppression or enhancement. Isotope-labelled internal standards (ILIS) were used to compensate the suppression or enhancement of signal intensities in the extracted samples. The method was validated using reference soil material (EUROSOIL 7) spiked with 50 μg/kg of each pesticide. The average recovery by PLE varied between 65.1% and 122.2% with RSDs of 1.7–23.4%. QuEChERS provided better recoveries for most of the pesticides, the extraction recovery ranging from 79.4% to 113.3% with RSDs of 1.0–12.2%. Limits of quantification for all target compounds were within a range of 0.1–2.9 µg/kg.     

Recently developed GC/MS and LC/MS methods for determining NSAIDs in water samples

Pharmaceuticals have become major targets in environmental chemistry due to their presence in aquatic environments (following incomplete removal in wastewater treatment or point-source contaminations), threat to drinking water sources and concern about their possible effects to wildlife and humans. Recently several methods have been developed for the determination of drugs and their metabolites in the lower nanogram per litre range, most of them using solid-phase extraction (SPE) or solid-phase microextraction (SPME), derivatisation and finally gas chromatography mass spectrometry (GC-MS), gas chromatography tandem mass spectrometry (GC-MS/MS) and liquid chromatography electrospray tandem mass spectrometry (LC-ES/MS/MS). Due to the elevated polarity of non-steroidal anti-inflamatory drugs (NSAIDs), analytical techniques based on either liquid chromatography coupled to mass spectrometry (LC-MS) and gas chromatography coupled to mass spectrometry (GC-MS) after a previous derivatisation step are essential. The most advanced aspects of current GC-MS, GC-MS/MS and LC-MS/MS methodologies for NSAID analysis are presented.     

LC-ESI/MS/MS method for rapid screening and confirmation of 44 exogenous anabolic steroids in human urine

A sensitive and rapid method based on liquid chromatography-triple-quadrupole tandem mass spectrometry (LC-MS/MS) with electrospray ionization (ESI) has been developed and validated for the screening and confirmation of 44 exogenous anabolic steroids (29 parent steroids and 15 metabolites) in human urine. The method involves an enzymatic hydrolysis, liquid-liquid extraction, and detection by LC-MS/MS. A triple-quadrupole mass spectrometer was operated in positive ESI mode with selected reaction monitoring (SRM) mode for the screening and product ion scan mode for the confirmation. The protonated molecular ions were used as precursor ions for the SRM analysis and product ion scan. The intraday and interday precisions of the target analytes at concentrations of the minimum required performance levels for the screening were 2-14% and 2-15%, respectively. The limits of detection for the screening and confirmation method were 0.1-10 ng/mL and 0.2-10 ng/mL, respectively, for 44 steroids. This method was successfully applied to analysis of urine samples from suspected anabolic steroid abusers.     

Analytical method for the determination of trace levels of steroid hormones and corticosteroids in soil, based on PLE/SPE/LC-MS/MS

The aim of this study was to develop an efficient, sensitive and reliable analytical method for the determination of traces of steroid hormones (including oestrogen, androgens and progestagens) and corticosteroids in soil. A method of sample preparation involving pressurized liquid extraction (PLE) and solid-phase extraction (SPE) was developed for the determination of six steroids and five corticosteroids in soils, followed by analysis by liquid chromatography-tandem mass spectrometry. The conditions employed for PLE involved acetone/methanol (50:50) as the extracting solvent, a temperature of 80 °C, two cycles and a static time of 5 min. The extraction was followed by a SPE clean-up based on a polymeric phase. With use of protocol, a residual matrix effect was, however, highlighted. The limit of detection in soil was 0.08–0.89 ng/g for steroids and 0.09–2.84 ng/g for corticosteroids.     

A single run LC-MS/MS method for phospholipidomics

Liquid chromatography coupled to tandem mass spectrometry has been compared to shotgun analysis with the objective of finding the best compromise for a single run analysis of whole cell phospholipids. Hydrophilic interaction liquid chromatography (HILIC), normal phase (NP), and reversed phase (RP) liquid chromatography were evaluated with reference phospholipids belonging to phosphatidic acid (PA), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylinositol (PI), and phosphatidylserine (PS) classes. NP-HPLC- and RP-HPLC-ESI-MS/MS were applied to yeast phospholipidome analysis, using a wild-type strain and two strains defective for acyltransferases that are known to be involved in de novo phospholipid synthesis or phospholipid remodeling. The MRM mode was used for relative quantitation of individual compounds based on reference phospholipids bearing fatty acid chains with an odd number of carbon atoms. Combined LC-MS/MS was found superior to shotgun analysis, leading to a larger number of quantified species than shotgun analysis. Finally, RP-HPLC-MS/MS was the preferred method for its higher selectivity, robustness, and better repeatability.     

Analyses of benzodiazepines and their metabolites in various biological matrices by LC-MS(/MS)

Benzodiazepines are among the most frequently prescribed drugs due to their sedative, hypnotic, anxiolytic, muscle relaxant and antiepileptic properties. Because of the high consumption of benzodiazepines worldwide, this class of drugs and their metabolites are frequently present in both clinical and forensic cases. For these reasons, the analysis of benzodiazepines and their metabolites in biological fluids is of great interest to clinicians and forensic toxicologists. This paper reviews procedures for multi-analyte single-stage (LC-MS) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) using different mass analyzers for the screening, identification and/or quantification of drugs, poisons and/or their metabolites in blood, plasma, serum or urine published since 2001. Basic information about the biosamples assayed, work-up, LC column, mobile phase, ionization type, mass spectral detection mode, matrix effects and validation data for each procedure is summarized. The feasibility of using LC-MS(/MS) techniques to identify and quantify benzodiazepines and their metabolites is also discussed.     

Quantification of pseudoephedrine in human plasma by LC-MS/MS using mosapride as internal standard

A simple, sensitive and rapid high-performance liquid chromatography/positive ion electrospray tandem mass spectrometry (LC-MS/MS) method was developed and validated for the quantification of pseudoephedrine in human plasma using mosapride as internal standard. Following solid-phase extraction, the analytes were separated using an isocratic mobile phase on a reverse-phase column and analyzed by MS/MS in the multiple-reaction monitoring mode using the respective [M + H](+) ions, m/z 166/148 for pseuoephedrine and m/z 422/198 for the IS. The method exhibited a linear dynamic range of 2-1000 ng/mL pseudoephedrine in human plasma. The lower limit of quantification was 2 ng/mL with a relative standard deviation of less than 9% for pseudoephedrine. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. The total chromatographic run time of 2 min for each sample made it possible to analyze more than 400 human plasma samples per day. The validated method has been successfully used to analyze human plasma samples for application in pharmacokinetic, bioavailability or bioequivalence studies.     

In electrospray ionization source hydrogen/deuterium exchange LC-MS and LC-MS/MS for characterization of metabolites

A new method is described for performing hydrogen/deuterium (H/D) exchange in an electrospray ionization (ESI) source. The use of liquid chromatography (LC)-mass spectrometer equipped with an ESI source and deuterium oxide (D2O) as the sheath liquid allows H/D exchange experiments to be performed on-line. This directly provides information for determining the number and position of exchangeable hydrogens, aiding in the elucidation of the structures of drug metabolites. To demonstrate the utility of this method, LC-mass spectrometry (MS) and LC-MS/MS experiments were performed using either H2O or D2O as sheath liquid on a matrix metalloprotease (MMP) inhibitor (PD 0200126) and its metabolites. Examination of the mass shift of the deuteriated molecule from that of the protonated molecule allowed the number of exchangeable protons to be determined. Interpretation of the production-spectra helped to determine the location of the exchanged protons and assisted in the assignment of the site(s) of modification for each metabolite.     

Development of a high performance liquid chromatography method and a liquid chromatography-tandem mass spectrometry method with pressurized liquid extraction for simultaneous quantification and confirmation of cyromazine, melamine and its metabolites in foods of animal origin

Simple and sensitive methods have been developed for simultaneous detection of cyromazine, melamine and their metabolites (ammeline, ammelide and cyanuric acid) in samples of animal origins. These include a high performance liquid chromatography (HPLC) method and a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method and are useful in regular monitoring and in toxicity studies of these molecules. Representative samples used in this study include muscles and livers of swine, bovine, sheep and chicken, kidneys of swine, bovine and sheep, and milk powder. A new sample preparation procedure with pressurized liquid extraction (PLE) at 1400psi and 70°C was investigated. Quantification of these five compounds by HPLC was achieved using an APS-2 column with UV detection at 230 nm. Limit of detection (LOD) was at 10 μgkg(-1), and limit of quantification (LOQ) was at 40 μgkg(-1). Recoveries of the five analytes in spiked samples ranged from 72.2% to 115.4% with RSD less than 12%. Confirmatory analysis of the analytes was performed using LC-MS/MS in selected reaction monitoring (SRM) mode. The LOD and LOQ were 5 μgkg(-1) and 15 μgkg(-1), respectively. This is the first simultaneous analysis of cyromazine, melamine, ammeline, ammelide and cyanuric acid residues in complex tissue samples using PLE and HPLC. It is expected that these methods will find many practical applications in evaluating the safety of cyromazine, melamine and their metabolites.     

Glycidyl fatty acid esters in food by LC-MS/MS: method development

An improved method based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the analysis of glycidyl fatty acid esters in oils was developed. The method incorporates stable isotope dilution analysis (SIDA) for quantifying the five target analytes: glycidyl esters of palmitic (C16:0), stearic (C18:0), oleic (C18:1), linoleic (C18:2) and linolenic acid (C18:3). For the analysis, 10 mg sample of edible oil or fat is dissolved in acetone, spiked with deuterium labelled analogs of glycidyl esters and purified by a two-step chromatography on C18 and normal silica solid phase extraction (SPE) cartridges using methanol and 5% ethyl acetate in hexane, respectively. If the concentration of analytes is expected to be below 0.5 mg/kg, 0.5 g sample of oil is pre-concentrated first using a silica column. The dried final extract is re-dissolved in 250 μL of a mixture of methanol/isopropanol (1:1, v/v), 15 μL is injected on the analytical C18 LC column and analytes are eluted with 100% methanol. Detection of target glycidyl fatty acid esters is accomplished by LC-MS/MS using positive ion atmospheric pressure chemical ionization operating in Multiple Reaction Monitoring mode monitoring 2 ion transitions for each analyte. The method was tested on replicates of a virgin olive oil which was free of glycidyl esters. The method detection limit was calculated to be in the range of 70-150 μg/kg for each analyte using 10 mg sample and 1-3 μg/kg using 0.5 g sample of oil. Average recoveries of 5 glycidyl esters spiked at 10, 1 and 0.1 mg/kg were in the range 84% to 108%. The major advantage of our method is use of SIDA for all analytes using commercially available internal standards and detection limits that are lower by a factor of 5-10 from published methods when 0.5 g sample of oil is used. Additionally, MS/MS mass chromatograms offer greater specificity than liquid chromatography-mass spectrometry operated in selected ion monitoring mode. The method will be applied to the survey of glycidyl fatty acid esters in food products on the Canadian market.     

超声萃取/液相色谱-串联质谱法测定染整助剂中的二硬脂基二甲基氯化铵

建立了超声辅助萃取/液相色谱-串联质谱( LC - MS/MS)测定染整助剂中二硬脂基二甲基氯化铵(DSDMAC)的方法.将样品以甲醇为萃取溶剂进行超声萃取,萃取液经过滤后采用LC-MS/MS测定.采用电喷雾离子源,定性离子对为m/z 550.5/298.3、522.5/298.3、522.5/270.3、494.5/...     

LC-MS/MS fast analysis of androgenic steroids in urine

The liquid chromatography-tandem mass spectrometry method (LC-MS/MS) was developed and validated to detect androgenic steroids: trenbolone, nortestosterone, boldenone, methylboldenone, testosterone, methyltestosterone, 17β-1-testosterone and their metabolites in bovine urine. Sample preparation before LC-MS/MS analysis involved an enzymatic hydrolysis with glucuronidase AS-HP, isolation of free hormones from urine on C(18) SPE column and purification of the extract using liquid-liquid extraction with n-pentane and SPE NH(2) column. For the chromatographic separation of steroids, the Poroshell 120-EC C18 column (150?×?2.1 mm, 2.7 μm) was used. Mass spectrometric measurement was achieved using the API 4000 triple quadrupole (QqQ) instrument with a TurboIon-Spray source operating in positive electrospray ionization mode. The procedure was validated according to the Decision 2002/657/EC. Recovery ranged from 76.5% to 118.9% for all examined compounds. The repeatability was below 20% and reproducibility did not exceed the 25%. The linearity was good for all analytes in the whole range of tested concentrations, as proved by the correlation coefficients greater than 0.99. The decision limit (CCα) ranged from 0.10 to 0.17 μg L(-1) for all analytes, whereas the detection capability (CCβ) ranged from 0.17 to 0.29 μg L(-1). The application of an innovative Poroshell column allowed for very good chromatographic separation of steroids with a much shorter time of analysis.     

LC-MS/MS Peptide Mapping with Automated Data Processing for Routine Profiling of N-Glycans in Immunoglobulins

Protein N-Glycan analysis is traditionally performed by high pH anion exchange chromatography (HPAEC), reversed phase liquid chromatography (RPLC), or hydrophilic interaction liquid chromatography (HILIC) on fluorescence-labeled glycans enzymatically released from the glycoprotein. These methods require time-consuming sample preparations and do not provide site-specific glycosylation information. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) peptide mapping is frequently used for protein structural characterization and, as a bonus, can potentially provide glycan profile on each individual glycosylation site. In this work, a recently developed glycopeptide fragmentation model was used for automated identification, based on their MS/MS, of N-glycopeptides from proteolytic digestion of monoclonal antibodies (mAbs). Experimental conditions were optimized to achieve accurate profiling of glycoforms. Glycan profiles obtained from LC-MS/MS peptide mapping were compared with those obtained from HPAEC, RPLC, and HILIC analyses of released glycans for several mAb molecules. Accuracy, reproducibility, and linearity of the LC-MS/MS peptide mapping method for glycan profiling were evaluated. The LC-MS/MS peptide mapping method with fully automated data analysis requires less sample preparation, provides site-specific information, and may serve as an alternative method for routine profiling of N-glycans on immunoglobulins as well as other glycoproteins with simple N-glycans.

Detection and quantification of benzodiazepines and Z-drugs in human whole blood, plasma, and serum samples as part of a comprehensive multi-analyte LC-MS/MS approach

For the first time, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) multi-analyte approach based on a simple liquid–liquid extraction was developed and validated for fast target screening and quantification of benzodiazepines and Z-drugs in case of driving ability and crime responsibility in the three most important biosamples whole blood, plasma, and serum. Whole blood, plasma, and serum (500 μL each) were extracted twice at pH 7.4 and at pH 10 with ether/ethyl acetate (1:1). Separation, detection, and quantification were performed using LC-MS/MS with electrospray ionization in positive mode. The method was validated with respect to selectivity, ion suppression/enhancement of co-eluting analytes, matrix effects, recovery, process efficiency, accuracy and precision, stabilities, and limits of detection and quantification. For accuracy and precision, full calibration was performed with ranges from subtherapeutic to toxic concentrations. The presented LC-MS/MS approach as part of a universal multi-analyte concept for over 100 drugs was applicable for selective detection as well as accurate and precise quantification in whole blood, plasma, and serum. The approach was selective, sensitive, accurate, and precise for 16 of the 19 tested drugs in whole blood, 18 in plasma, and 17 in serum. Only semiquantitative results could be obtained for zopiclone because of its instability in all tested biosamples.     

Progress and Challenges in Quantifying Carbonyl-Metabolomic Phenomes with LC-MS/MS

Carbonyl-containing metabolites widely exist in biological samples and have important physiological functions. Thus, accurate and sensitive quantitative analysis of carbonyl-containing metabolites is crucial to provide insight into metabolic pathways as well as disease mechanisms. Although reversed phase liquid chromatography electrospray ionization mass spectrometry (RPLC-ESI-MS) is widely used due to the powerful separation capability of RPLC and high specificity and sensitivity of MS, but it is often challenging to directly analyze carbonyl-containing metabolites using RPLC-ESI-MS due to the poor ionization efficiency of neutral carbonyl groups in ESI. Modification of carbonyl-containing metabolites by a chemical derivatization strategy can overcome the obstacle of sensitivity; however, it is insufficient to achieve accurate quantification due to instrument drift and matrix effects. The emergence of stable isotope-coded derivatization (ICD) provides a good solution to the problems encountered above. Thus, LC-MS methods that utilize ICD have been applied in metabolomics including quantitative targeted analysis and untargeted profiling analysis. In addition, ICD makes multiplex or multichannel submetabolome analysis possible, which not only reduces instrument running time but also avoids the variation of MS response. In this review, representative derivatization reagents and typical applications in absolute quantification and submetabolome profiling are discussed to highlight the superiority of the ICD strategy for detection of carbonyl-containing metabolites.     

LC-MS/MS profiling for detection of endogenous steroids and prostaglandins in tissue samples

Roles of steroid hormones, and compounds that can influence their levels in cells, are of increasing interest in e.g. cancer research, partly because resistance to hormone therapies often complicates treatment. To elucidate the processes involved, the hormones and related compounds need to be accurately measured. Reversed-phase liquid chromatography with dynamic multiple reaction monitoring mass spectrometric detection in electrospray mode is capable of providing such measurements. Therefore, LC-MS/MS was developed for sensitive, selective analysis of 11 steroid hormones, cholesterol and two prostaglandins. The effects of the tissue matrix, and solid-phase extraction (SPE) sample clean-up, on the LC-MS/MS signals of the hormones were also investigated. The results show that the developed LC-MS/MS method, following SPE clean-up to reduce matrix interference, can detect selected steroids in extracts of mouse tissues. The method provides linear measurements of the steroids at concentrations up to few ng/μL, and limits of detection in the range 0.03-0.2 pg/μL (for some compounds lower than those of previously reported methods).     

Development and application of a new on-line SPE system combined with LC-MS/MS detection for high throughput direct analysis of pharmaceutical compounds in plasma

A technique using a fully automated on-line solid phase extraction (SPE) system (Symbiosis, Spark Holland) combined with liquid chromatography (LC)-mass spectrometry (MS/MS) has been investigated for fast bioanalytical method development, method validation and sample analysis using both conventional C18 and monolithic columns. Online SPE LC-MS/MS methods were developed in the automated mode for the quantification of model compounds (propranolol and diclofenac) directly in rat plasma. Accuracy and precision using online SPE LC-MS/MS with conventional C18 and monolithic columns were in the range of 88-111% and 0.5-14%, respectively. Total analysis cycle time of 4 min per sample was demonstrated using the C18 column. Monolithic column allowed for 2 min total cycle time without compromising the quality and validation criteria of the method. Direct plasma sample injection without on-line SPE resulted in poor accuracy and precision in the range of 41-108% and 3-81%. Furthermore, the increase in back pressure resulted in column damage after the injection of only 60 samples.     

Application of LC-MS/MS method for the in vivo metabolite determination of oleuropein after intravenous administration to rat

A highly sensitive, specific and simple LC‐MS/MS method was developed to investigate in vivo bio‐transformation of oleuropein in rat. Rat urine samples collected after the intravenous administrations were determined using liquid chromatography coupled to tandem mass spectrometry with electrospray ionization in the negative‐ion mode. The assay procedure involves a simple liquid–liquid extraction of parent oleuropein and the metabolite from rat urine with ethyl acetate. Chromatographic separation was operated with 0.1% formic acid aqueous and methanol in gradient program at a flow rate of 0.80 mL/min on an RP‐C₁₈ column with a total run time of 30 min. This method has been successfully applied to simultaneous determination of oleuropein and its metabolite in rat urine. Oxygenation was found to be the major metabolic pathway of the oleuropein in rat after intravenous administration. Copyright © 2011 John Wiley & Sons, Ltd.     

Validation of a novel LC-MS/MS method for the quantitation of colistin A and B in human plasma

A liquid chromatography/tandem mass spectrometry (LC-MS/MS) method, characterized by complete automation and high-throughput, was developed for the determination of colistin A and B in human plasma. All sample preparation procedures were performed by using 2.2 mL 96-deep-well plates, whereas robotic liquid-handling workstations were utilized for all liquid transfer steps, including solid-phase extraction (SPE). The whole preparation procedure was very rapid, whereas the method had a very short chromatographic run time of just 2 min. Sample analysis was performed by reversed phase LC-MS/MS, with positive electrospray ionization, using multiple reaction monitoring. The absence of available purified colistin A and B standards led to the development of a novel LC method with evaporative light-scattering detector for the determination of their stoichiometries in the standard mixture, along with its purity. The proposed bioanalytical method was fully validated and it was proven to be selective, accurate, precise, reproducible and suitable for the determination of colistin A and B in human plasma. It was applied successfully to a pharmacokinetic study for the determination of both analytes in samples of patients.