Characterization of nonyl phenol ethoxylates in sewage treatment plants by combined precursor ion scanning and multiple reaction monitoring.

A novel rapid screening technique for the profiling of nonyl phenol ethoxylates (NPEs) in sewage treatment plant (STP) influent/effluent was developed using flow injection with atmospheric pressure ionization coupled to mass spectrometric analysis by combined precursor ion scanning and multiple reaction monitoring. The technique allows for total NPE concentrations as low as 50 parts-per-trillion to be detected in STP samples.     

Comparison of targeted peptide quantification assays for reductive dehalogenases by selective reaction monitoring (SRM) and precursor reaction monitoring (PRM)

Targeted absolute protein quantification yields valuable information about physiological adaptation of organisms and is thereby of high interest. Especially for this purpose, two proteomic mass spectrometry-based techniques namely selective reaction monitoring (SRM) and precursor reaction monitoring (PRM) are commonly applied. The objective of this study was to establish an optimal quantification assay for proteins with the focus on those involved in housekeeping functions and putative reductive dehalogenase proteins from the strictly anaerobic bacterium Dehalococcoides mccartyi strain CBDB1. This microbe is small and slow-growing; hence, it provides little biomass for comprehensive proteomic analysis. We therefore compared SRM and PRM techniques. Eleven peptides were successfully quantified by both methods. In addition, six peptides were solely quantified by SRM and four by PRM, respectively. Peptides were spiked into a background of Escherichia coli lysate and the majority of peptides were quantifiable down to 500 amol absolute on column by both methods. Peptide quantification in CBDB1 lysate resulted in the detection of 15 peptides using SRM and 14 peptides with the PRM assay. Resulting quantification of five dehalogenases revealed copy numbers of <10 to 115 protein molecules per cell indicating clear differences in abundance of RdhA proteins during growth on hexachlorobenzene. Our results indicated that both methods show comparable sensitivity and that the combination of the mass spectrometry assays resulted in higher peptide coverage and thus more reliable protein quantification.

Rapid and sensitive identification of ricin in environmental samples based on lactamyl agarose beads using LC‐MS/MS (MRM)

Ricin, a plant‐derived toxin extracted from the seeds of Ricinus communis (castor bean plant), is one of the most toxic proteins known. Ricin's high toxicity, widespread availability, and ease of its extraction make it a potential agent for bioterrorist attacks. Most ricin detection methods are based on immunoassays. These methods may suffer from low efficiency in matrices containing interfering substances, or from false positive results due to antibody cross reactivity, with highly homologous proteins. In this study, we have developed a simple, rapid, sensitive, and selective mass spectrometry assay, for the identification of ricin in complex environmental samples. This assay involves three main stages: (a) Ricin affinity capture by commercial lactamyl‐agarose (LA) beads. (b) Tryptic digestion. (c) LC‐MS/MS (MRM) analysis of tryptic fragments. The assay was validated using 60 diverse environmental samples such as soil, asphalt, and vegetation, taken from various geographic regions. The assay's selectivity was established in the presence of high concentrations of competing lectin interferences. Based on our findings, we have defined strict criteria for unambiguous identification of ricin. Our novel method, which combines affinity capture beads followed by MRM‐based analysis, enabled the identification of 1 ppb ricin spiked into complex environmental matrices. This methodology has the potential to be extended for the identification of ricin in body fluids from individuals exposed (deliberately or accidentally) to the toxin, contaminated food or for the detection of the entire family of RIP‐II toxins, by applying multiplex format.     

Biopolymer sequencing using a triple quadrupole mass spectrometer in the ESI nozzle-skimmer/precursor ion MS/MS mode

A variety of model biopolymers, including oligonucleotides, oligosaccharides and a synthetic pharmaceutical agent, were sequenced using a triple quadrupole mass spectrometer equipped with an electrospray source and operated in a scan mode referred to as pseudo-MS3. This scan mode consists of three steps: (1) in-source collision-induced dissociation (CID) in the nozzle-skimmer (NS) region, (2) scanning of the fragment ions into the collision cell for further CID, and (3) passing of the secondary fragment ions through the final mass filter at a preselected mass, generally corresponding to the mass of a terminal sequence ion for the biopolymer. The mass spectra are recorded in the precursor ion MS/MS mode where ion selection and detection occur at the third stage of the triple quadrupole but the scan function is determined by the first stage. The advantages and limitations in using this pseudo-MS3 NS/precursor ion MS/MS scan mode for biopolymer sequencing are discussed.     

Optimization of multiple reaction monitoring mode for the trace analysis of veterinary sulfonamides by LC-MS/MS

One of the oldest groups of veterinary chemotherapeutic agents, sulfonamides have been widely used for more than 50 years, thanks to their low cost and their broad spectrum of activity in preventing or treating bacterial infections. Nowadays, those compounds are regularly detected in a wide variety of environmental samples, including natural waters, sediments and soils. Since the environmental concentrations of sulfonamides are usually very low and their occurrence multicomponental, their determination in these matrices still pose significant analytical problems. The present paper describes the optimization of ESI-MS/MS parameters and the chromatographic separation of 12 sulfonamides commonly used in veterinary medicine. The methodology developed in this study, unlike many others, satisfied the requirements of EU Commission Decision 2002/657/EC, which defines the criteria for both screening and confirmatory methods with respect to drug residues on the basis of identification points. Each MRM transition was tested not only for the qualitative but also for the quantitative analysis of sulfonamides. The method was validated for its analytical performance parameters and applied to the determination of those compounds in soil samples.     

Identification and characterization of stressed degradation products of metoprolol using LC/Q-TOF-ESI-MS/MS and MS(n) experiments

A rapid, specific and reliable isocratic high-performance liquid chromatography combined with quadrupole time-of-flight electrospray ionization tandem mass spectrometry (LC/Q-TOF-ESI-MS/MS) method has been developed and validated for the identification and characterization of stressed degradation products of metoprolol. Metoprolol, an anti-hypertensive drug, was subjected to hydrolysis (acidic, alkaline and neutral), oxidation, photolysis and thermal stress, as per ICH-specified conditions. The drug showed extensive degradation under oxidative and hydrolysis (acid and base) stress conditions. However, it was stable to thermal, neutral and photolysis stress conditions. A total of 14 degradation products were observed and the chromatographic separation of the drug and its degradation products was achieved on a C(18) column (4.6 × 250 mm, 5 μm). To characterize degradation products, initially the mass spectral fragmentation pathway of the drug was established with the help of MS/MS, MS(n) and accurate mass measurements. Similarly, fragmentation pattern and accurate masses of the degradation products were established by subjecting them to LC-MS/QTOF analysis. Structure elucidation of degradation products was achieved by comparing their fragmentation pattern with that of the drug. The degradation products DP(2) (m/z 153) and DP(14) (m/z 236) were matched with impurity B, listed in European Pharmacopoeia and British Pharmacopoeia, and impurity I, respectively. The LC-MS method was validated with respect to specificity, linearity, accuracy and precision.     

Targeted profiling and relative quantification of benzoyl diterpene alkaloids in Aconitum roots by using LC–MS/MS with precursor ion scan

Benzoyl aconite alkaloids have myocardial protective effects at a low dose and produce toxic effects at high dose. Due to lack of enough reference compounds, most of the benzoyl alkaloids had few concerns, except the typical ones, i.e. aconitine, mesaconitine, and hypaconitine. To rapidly screen out and quantify benzoyl alkaloids, a high performance liquid chromatography combined with tandem mass spectrometry was proposed based on precursor ion scanning mode. First, a diagnostic ion at m/z 105 corresponding to benzoyl group was observed by using tandem mass spectrometry, which could be used for the rapid identification of benzoyl alkaloids. The targeted screening of these alkaloids was then conducted by using precursor ion scan of characteristic ion at m/z 105. Shengfuzi (the lateral root of A. carmichaelii) was taken as example, and 24 benzoyl‐containing alkaloids were identified. The six major alkaloids including aconitine, mesaconitine, hypaconitine, benzoylaconine, benzoylmesaconine, and benzoylhypaconine were determined in the precursor ion scan mode by the standard curve method. Reliable linearity, sensitivity, precision, accuracy, and repeatability were obtained and validated. Then the relative response factors between these six analytes were calculated, which were not more than two times using any alkaloid as reference. Thus, the other 18 alkaloids lacking reference compounds were relatively quantified. This approach provides a useful tool for rapid identification and quantitative analysis of toxic benzoyl alkaloids, and also an efficient method for the safety assessment of Aconitum roots.     

Qualitative and Quantitative Analysis of Ejiao-Related Animal Gelatins through Peptide Markers Using LC-QTOF-MS/MS and Scheduled Multiple Reaction Monitoring (MRM) by LC-QQQ-MS/MS

Donkey-hide gelatin, also called Ejiao (colla corii asini), is commonly used as a food health supplement and valuable Chinese medicine. Its growing popular demand and short supply make it a target for fraud, and many other animal gelatins can be found as adulterants. Authentication remains a quality concern. Peptide markers were developed by searching the protein database. However, donkeys and horses share the same database, and there is no specific marker for donkeys. Here, solutions are sought following a database-independent strategy. The peptide profiles of authentic samples of different animal gelatins were compared using LC-QTOF-MS/MS. Fourteen specific markers, including four donkey-specific, one horse-specific, three cattle-specific, and six pig-specific peptides, were successfully found. As these donkey-specific peptides are not included in the current proteomics database, their sequences were determined by de novo sequencing. A quantitative LC-QQQ multiple reaction monitoring (MRM) method was further developed to achieve highly sensitive and selective analysis. The specificity and applicability of these markers were confirmed by testing multiple authentic samples and 110 batches of commercial Ejiao products, 57 of which were found to be unqualified. These results suggest that these markers are specific and accurate for authentication purposes.     

Fragmentation pathways of organoarsenical compounds by electrospray ion trap multiple mass spectrometry (MS6)

With its detection limit well below 30 pg microl(-1) LC-MS-MS has become a sensitive and thus popular analytical technique for organoarsenical compounds. Collision induced dissociation (CID) is a valuable tool for speciation and facilitates a positive identification of the species detected. However, it is not straightforward to understand the fragmentation pathways of organoarsenical compounds when only CID-MS-MS data is available. In the present paper we have investigated multiple mass spectrometry (MSn, n=1, 2, 3, 4, 5, 6) with electrospray CID fragmentation for a number of organoarsenical compounds likely to occur in the environment. The investigated compounds were tetramethylarsonium, trimethylarsinoxide, monomethylarsonic acid, dimethylarsinic acid, arsenobetaine, arsenocholine, and dimethylarsinoylethanol. By CID of (protonated) organoarsenical cations mostly even-electron fragments are produced after neutral loss processes such as elimination of H2, H2O, CH4, C2H2, C2H4, C2H6, HCHO, CH3OH, C2H5OH, C2H4O, and CH2CO. However, abundant odd-electron fragments are also formed after elimination of radical species. Evidence for reduction of As(V) to As(III) as a driving force in the odd-electron ion formation is obtained.     

Optimized precursor ion selection for labile ions in a linear ion trap mass spectrometer and its impact on quantification using selected reaction monitoring

The fragmentation of fragile ions during the application of an isolation waveform for precursor ion selection and the resulting loss of isolated ion intensity is well‐known in ion trap mass spectrometry (ITMS). To obtain adequate ion intensity in the selected reaction monitoring (SRM) of fragile precursor ions, a wider ion isolation width is required. However, the increased isolation width significantly diminishes the selectivity of the channels chosen for SRM, which is a serious problem for samples with complex matrices. The sensitive and selective quantification of many lipid molecules, including ceramides from real biological samples, using a linear ion trap mass spectrometer is also hindered by the same problem because of the ease of water loss from protonated ceramide ions. In this study, a method for the reliable quantification of ceramides using SRM with near unity precursor ion isolation has been developed for ITMS by utilizing alternative precursor ions generated by in‐source dissociation. The selected precursor ions allow the isolation of ions with unit mass width and the selective analysis of ceramides using SRM with negligible loss of sensitivity. The quantification of C18:0‐, C24:0‐ and C24:1‐ceramides using the present method shows excellent linearity over the concentration ranges from 6 to 100, 25 to 1000 and 25 to 1000 nM, respectively. The limits of detection of C18:0‐, C24:0‐ and C24:1‐ceramides were 0.25, 0.25 and 5 fmol, respectively. The developed method was successfully applied to quantify ceramides in fetal bovine serum. Copyright © 2014 John Wiley & Sons, Ltd.     

Hair-based rapid analyses for multiple drugs in forensics and doping: application of dynamic multiple reaction monitoring with LC-MS/MS

Background

Considerable efforts are being extended to develop more effective methods to detect drugs in forensic science for applications such as preventing doping in sport. The aim of this study was to develop a sensitive and accurate method for analytes of forensic and toxicological nature in human hair at sub-pg levels.

Results

The hair test covers a range of different classes of drugs and metabolites of forensic and toxicological nature including selected anabolic steroids, cocaine, amphetamines, cannabinoids, opiates, bronchodilators, phencyclidine and ketamine. For extraction purposes, the hair samples were decontaminated using dichloromethane, ground and treated with 1 M sodium hydroxide and neutralised with hydrochloric acid and phosphate buffer and the homogenate was later extracted with hexane using liquid-liquid extraction (LLE). Following extraction from hair samples, drug-screening employed liquid chromatography coupled to tandem mass spectrometric (LC-MS/MS) analysis using dynamic multiple reaction monitoring (DYN-MRM) method using proprietary software. The screening method (for?>?200 drugs/metabolites) was calibrated with a tailored drug mixture and was validated for 20 selected drugs for this study. Using standard additions to hair sample extracts, validation was in line with FDA guidance. A Zorbax Eclipse plus C18 (2.1 mm internal diameter × 100 mm length × 1.8 μm particle size) column was used for analysis. Total instrument run time was 8 minutes with no noted matrix interferences. The LOD of compounds ranged between 0.05-0.5 pg/mg of hair. 233 human hair samples were screened using this new method and samples were confirmed positive for 20 different drugs, mainly steroids and drugs of abuse.

Conclusions

This is the first report of the application of this proprietary system to investigate the presence of drugs in human hair samples. The method is selective, sensitive and robust for the screening and confirmation of multiple drugs in a single analysis and has potential as a very useful tool for the analysis of large array of controlled substances and drugs of abuse.

     

Screening of methylenedioxyamphetamine‐ and piperazine‐derived designer drugs in urine by LC–MS/MS using neutral loss and precursor ion scan

This study describes a method for the screening of methylenedioxyamphetamine‐ and piperazine‐derived compounds in urine by liquid chromatography‐tandem mass spectrometry. These substances, characterized by possessing common moieties, are screened using precursor ion and neutral loss scan mode and then quantified in multiple reaction monitoring acquisition mode. Based on the product‐ion spectra of different known molecules, chosen as ‘model’, characteristic neutral losses and product ions were selected: piperazines were detected in precursor ion scan of m/z 44 and neutral loss of 43 and 86 while amphetamines in precursor ion scan of m/z 133, 135 and 163. The applicability of the screening approach was studied in blank urine spiked with selected analytes and processed by solid‐phase extraction. Linearity, matrix effect, precision, accuracy, limits of detection and limits of quantification were evaluated both for the screening and the quantification methods. The ability of the screening method to provide semi‐quantitative data was demonstrated. This method appears to be a useful tool for the identification of designer drugs derived from piperazines or methylenedioxyamphetamines and can be potentially applied to other drug classes. Copyright © 2013 John Wiley & Sons, Ltd.     

Rapid screening and characterization of drug metabolites using a multiple ion monitoring-dependent MS/MS acquisition method on a hybrid triple quadrupole-linear ion trap mass spectrometer

A novel LC/MS/MS method that uses multiple ion monitoring (MIM) as a survey scan to trigger the acquisition of enhanced product ions (EPI) on a hybrid quadrupole-linear ion trap mass spectrometer (Q TRAP) was developed for drug metabolite identification. In the MIM experiment, multiple predicted metabolite ions were monitored in both Q1 and Q3. The collision energy in Q2 was set to a low value to minimize fragmentation. Results from analyzing ritonavir metabolites in rat hepatocytes demonstrate that MIM-EPI was capable of targeting a larger number of metabolites regardless of their fragmentation and retained sensitivity and duty cycle similar to multiple reaction monitoring (MRM)-EPI. MIM-based scanning methods were shown to be particularly useful in several applications. First, MIM-EPI enabled the sensitive detection and MS/MS acquisition of up to 100 predicted metabolites. Second, MIM-MRM-EPI was better than MRM-EPI in the analysis of metabolites that undergo either predictable or unpredictable fragmentation pathways. Finally, a combination of MIM-EPI and full-scan MS (EMS), as an alternative to EMS-EPI, was well suited for routine in vitro metabolite profiling. Overall, MIM-EPI significantly enhanced the metabolite identification capability of the hybrid triple quadrupole-linear ion trap LC/MS.     

Simultaneous selected reaction monitoring, MS/MS and MS3 quantitation for the analysis of pharmaceutical compounds in human plasma using chip-based infusion

An assay method with mass spectrometric detection was developed for the quantitative analysis of a pharmaceutical compound and its major metabolite in human plasma using chip-based infusion. Liquid-liquid extraction sample preparation was found to be essential to minimize matrix suppression and to achieve a limit of quantitation (LOQ) of 2.5 ng/mL using a 100 microL plasma aliquot. The potential for simultaneous quantitation in selected reaction monitoring (SRM), tandem mass spectrometry (MS/MS) (enhanced product ion), and MS(3) was investigated and found to be very beneficial in improving assay selectivity. A novel concept for monitoring quantitative assay performance using a SRM/MS(3) ratio is proposed.     

Structure elucidation of cyclic pyoverdins and examination of rearrangement reactions in MS/MS experiments by determination of exact product ion masses

Structure elucidation of naturally occurring linear and cyclic peptidic compounds can be complicated by rearrangement reactions induced upon collision activation (CA) when parts of the molecule migrate, suggesting incorrect substitution patterns. Such complex rearrangements are examined and discussed for two iron complexing compounds produced by the bacterial genus Pseudomonas (so-called pyoverdins). Various MS2- and MS3-product ion experiments were performed using a quadrupole-ion trap (QIT) at low resolution and a FT-ICR at high resolution allowing accurate mass determinations. The results of the multidimensional study confirm the proposed processes. On the basis of the series of tandem-MS experiments the structure of a new pyoverdin from a P. fluorescens strain [PVD(D47)] is deduced.