Pseudo Multiple Reaction Monitoring Research Articles

Removal of isobaric interference using pseudo-multiple reaction monitoring and energy-resolved mass spectrometry for the isotope dilution quantification of a tryptic peptide.

Energy-resolved mass spectrometry (ERMS) and an isotopically labelled internal standard were successfully combined to accurately quantify a tryptic peptide despite the presence of an isobaric interference. For this purpose, electrospray ionisation tandem mass spectrometry (ESI-MS/MS) experiments were conducted into an ion trap instrument using an unconventional 8m/z broadband isolation window, which encompassed both the tryptic peptide and its internal standard. Interference removal was assessed by determining an excitation voltage that was high enough to maintain a constant value for the analyte/internal standard peaks intensity ratio, thus ensuring accurate quantification even in the presence of isobaric contamination. Pseudo-multiple reaction monitoring (MRM) was employed above this excitation voltage to quantify the trypic peptide. The internal standard calibration model showed no lack of fit and exhibited a linear dynamic range from 0.5μM up to 2.5μM. The detection limit was 0.08 μM. The accuracy of the method was evaluated by quantifying the tryptic peptide of three reference samples intentionally contaminated with the isobaric interference. All the reference samples were accurately quantified with ∼1% deviation despite the isobaric contamination. Furthermore, we have demonstrated that this methodology can also be applied to quantify the isobaric peptide by standard additions down to 0.2μM. Finally, liquid chromatography ERMS (LC ERMS) experiments yielded similar results, suggesting the potential of the proposed methodology for analysing complex samples.

Accurate Detection of G-Series Agent Simulants by Pseudo-Multiple Reaction Monitoring on Miniature LIT-MS.

Rapid and accurate on-site detection of chemical warfare agents (CWAs) could defend military and civilian populations against current and emerging chemical weapons. With the development of ambient ionization and linear ion trap technology, the rapid and accurate quantitative determination method of CWAs based on direct ionization and multistage mass spectrometry has attracted widespread attention. In this study, a microliter electrospray ionization-miniature linear ion trap mass spectrometry (LIT-MS) instrument was designed and constructed, and the effects of quadrupole enhanced dipole resonance excitation on the resolution and sensitivity were investigated; consequently, the parameters of CWAs detection were optimized. Based on the broad time-frequency ion excitation technology, accurate multiple reaction monitoring (MRM) quantitative analysis of DMMP (G-series agent simulants, m/z 125 → m/z 93) was obtained. The linear correlation coefficient in the concentration range of 1 to 20 μg/mL could reach 99.02%, and the relative standard deviations (RSD) of continuous repeatability, interday repeatability, and intraday repeatability were all less than 10%. The results showed that the accurate pseudo-MRM detection method based on miniature linear ion trap mass spectrometry for CWAs detection was feasible.

CIL-ExPMRM: An Ultrasensitive Chemical Isotope Labeling Assisted Pseudo-MRM Platform to Accelerate Exposomic Suspect Screening.

Exposome is the future of next-generation environmental health to establish the association between environmental exposure and diseases. However, due to low concentrations of exposure chemicals, exposome has been hampered by lacking an effective analytical platform to characterize its composition. In this study, by combining the benefit of chemical isotope labeling and pseudo-multiple reaction monitoring (CIL-pseudo-MRM), we have developed one highly sensitive and high-throughput platform (CIL-ExPMRM) by isotope labeling urinary exposure biomarkers. Dansyl chloride (DnsCl), N-methylphenylethylamine (MPEA), and their isotope-labeled forms were used to derivatize polar hydroxyl and carboxyl compounds, respectively. We have programmed a series of scripts to optimize MRM transition parameters, curate the MRM database (>70,000 compounds), predict accurate retention time (RT), and automize dynamic MRMs. This was followed by an automated MRM peak assignment, peak alignment, and statistical analysis. A computational pipeline was eventually incorporated into a user-friendly website interface, named CIL-ExPMRM (http://www.exposomemrm.com/). The performance of this platform has been validated with a relatively low false positive rate (10.7%) across instrumental platforms. CIL-ExPMRM has systematically overcome key bottlenecks of exposome studies to some extent and outperforms previous methods due to its independence of MS/MS availability, accurate RT prediction, and collision energy optimization, as well as the ultrasensitivity and automated robust intensity-based quantification. Overall, CIL-ExPMRM has great potential to advance the exposomic studies based on urinary biomarkers.

Combination of Structure Databases, In Silico Fragmentation, and MS/MS Libraries for Untargeted Screening of Non-Volatile Migrants from Recycled High-Density Polyethylene Milk Bottles.

Chemical contamination is one of the major obstacles for mechanical recycling of plastics. In this article, we built and open-sourced an in-house MS/MS library containing more than 500 plastic-related chemicals and developed mspcompiler, an R package, for the compilation of various libraries. We then proposed a workflow to process untargeted screening data acquired by liquid chromatography high-resolution mass spectrometry. These tools were subsequently employed to data originating from recycled high-density polyethylene (rHDPE) obtained from milk bottles. A total of 83 compounds were identified, with 66 easily annotated by making use of our in-house MS/MS libraries and the mspcompiler R package. In silico fragmentation combined with data obtained from gas chromatography-mass spectrometry and lists of chemicals related to plastics were used to identify those remaining unknown. A pseudo-multiple reaction monitoring method was also applied to sensitively target and screen the identified chemicals in the samples. Quantification results demonstrated that a good sorting of postconsumer materials and a better recycling technology may be necessary for food contact applications. Removal or reduction of non-volatile substances, such as octocrylene and 2-ethylhexyl-4-methoxycinnamate, is still challenging but vital for the safe use of rHDPE as food contact materials.

Parallel Pseudo-MRM on the “brick” miniature mass spectrometer for high throughput multi-target screening

With increasingly improved analytical performances, miniature mass spectrometers are expandingly applied in more application scenarios. The capability of high throughput analysis of multi-targets is a crucial function of a miniature mass spectrometer, especially for in-situ applications. As a powerful method for target screening, multiple reaction monitoring (MRM) was conventionally realized on triple quadrupole (QQQ) mass spectrometers. Previously, pseudo-multiple reaction monitoring (pseudo-MRM) operation mode has been realized on a “brick” mass spectrometer equipped with a single ion trap. However, pseudo-MRM on an ion trap was realized through the tandem-in-time fashion, and the throughput of pseudo-MRM was still limited. As a continuous effort to boost performances of the “brick” miniature mass spectrometer, parallel pseudo-MRM mode was developed, and the throughput could be raised up to 10 times for multi-target screening. To achieve better isolation and collision induced dissociation (CID) efficiencies, the mass range of interest was divided into several segments based on optimized Mathieu q value. The fast screening of 10 different drugs was used as an illustrative example. Furthermore, parallel quantitative analysis was also demonstrated and verified using the internal standard calibration method.

Detection and differentiation of active and inactive isoforms of coagulation factors II, VII, IX, and X in prothrombin complex concentrate by mass spectrometry

PurposeProthrombin complex concentrates (PCCs) are plasma products containing a mixture of four inactive/proactive coagulation factors. The activated forms of human coagulation factors, like Thrombin (FIIa), Convertin (FVIIa), activated Christmas factor (FIXa) and the activated Stuart-Prower factor (FXa), are impurities in PCCs. Until now no valid assay exists to differentiate the non activated proform (inactive) from active coagulation factor isoforms in PCCs in one measurement. Therefore, the aim of this study was to establish a mass spectrometry (LC-MS/MS)-based assay to address this issue in the ready to use medicinal product. MethodsBottom-up proteomics combining double digestion (Glu-C & Lys-C) and LC-MS/MS, was used to differentiate the inactive and active forms of the coagulation factors Prothrombin (FII), Proconvertin (FVII), Christmas factor (FIX) and the Stuart-Prower-factor (FX) in PCCs. Results and conclusionsA targeted pseudo-multiple reaction monitoring (pMRM-LC-MS/MS)-assay was developed for the specific detection of four different coagulation factors in PCCs. Proteotypic peptides for the inactive/active isoforms (zymogen) of the four coagulation factors were identified and validated by the investigation of six investigational and one commercially available PCCs.In conclusion, the semi-quantitative determination and the distinction between the active and the inactive isoform of the respective coagulation factors were possible in one liquid chromatography tandem mass spectrometry (LC-MS/MS) run.

Development of a liquid chromatography-electrospray ionization tandem mass spectrometric method for the simultaneous analysis of free fatty acids.

Fatty acids (FAs) play important roles in several physiological and pathophysiological processes, functioning as both nonesterified free FAs (FFAs) and components of other lipid classes. Although many lipid classes are readily measured using liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS), the measurement of FFAs by this method is not straightforward because of inconsistent fragmentation behaviours. In this study, we describe a strategy to measure FFAs using conventional reverse-phase LC-ESI-MS/MS, without derivatization. The strategy combines three key methods: (i) an isocratic LC separation with a high organic solvent ratio, (ii) postcolumn base addition, and (iii) pseudo-multiple reaction monitoring. The method facilitates the measurement of ultra-long-chain FAs, the accumulation of which is a common biochemical abnormality in peroxisomal disorders. This study delivers a broad strategy that measures a wide spectrum of FFA species in complex biological samples.

Derivatization assisted LC-p-MRM-MS with high CID voltage for rapid analysis of brassinosteroids

In this study, a derivatization-assisted pseudo-multiple reaction monitoring with high CID voltage (HV-p-MRM) strategy was proposed for the analysis of brassinosteroids (BRs) by liquid chromatography-triple quadrupole mass spectrometry (LC-QqQ MS). The concept of the HV-p-MRM strategy was proposed on the basis of an assumption that the precursor ion of analyte is stable in collision cell and less prone to fragmentation at high CID voltage, while co-existing ions (impurity) of easy fragmentation can break down into smaller fragment ions. In such case, by increasing the CID voltage, the co-existing ions that are introduced due to the low resolution of the quadrupole 1 (Q1) can be filtered out by quadrupole 3 (Q3), while the stable precursor ion of analyte will pass through Q3, thereby that the signal-to-noise ratio (S/N) of the analysis can be improved. As a proof-of-concept study, BRs were derivatized with rhodamine B-boronic acid (RhB-BA) and then the derivatives were used to investigate their variations in MS signal, background noise, and S/N upon the CID voltage and MS scanning resolution. The results showed that S/N of these derivatives can be improved in HV-p-MRM mode. To further demonstrate the feasibility of HV-p-MRM strategy, a RhB-BA derivatization assisted LC-HV-p-MRM-MS method was developed for the analysis of BRs. Using this method, rapid and sensitive determination of BRs in different organs of rape flower was achieved. It can be expected that HV-p-MRM may be suitable for the analytes that are stable or can be converted into compounds of high stability in collision cell at high CID voltage.

Understanding the relative performance of SCAN, SIM, PMRM and MRM methods for quantifying polycyclic aromatic hydrocarbons in crude oil samples.

Polycyclic aromatic hydrocarbons (PAHs) present in oil spill samples are analyzed by gas chromatography/mass spectrometry (GC/MS) and gas chromatography/tandem mass spectrometry (GC/MS/MS) using four different methods: (1) full scan (SCAN), (2) selected ion monitoring (SIM), (3) multiple reaction monitoring (MRM), and (4) pseudo multiple reaction monitoring (PMRM). This study quantifies the relative performance of these methods. Novel experiments were designed to measure the signal-to-noise (S/N) ratios of all four methods. This was accomplished by spiking the crude oil with five deuterated PAHs (dPAHs) in two distinct ways: (1) varying the background noise by changing crude oil concentrations before spiking the samples with 1 ng/mL of dPAHs, and (2) varying the signal by spiking dPAHs concentrations of 0.5 and 5 ng/mL into a crude oil sample. The MRM method is the most selective and sensitive of the four methods. It also provides the lowest limit of detection (LOD) and limit of quantitation (LOQ). MRM is the optimal approach for quantifying PAHs in complex petroleum samples containing high levels of background noise. Also, our data show that the PAHs in complex oil spill samples can be quantified by MRM without using any complicated sample preparation steps. Based on our experimental data, the relative performance of the four methods used for quantifying PAHs in crude oil samples can be ranked as MRM > PMRM > SIM > SCAN.

Determination of kaurenoic acid in rat plasma using UPLC-MS/MS and its application to a pharmacokinetic study

Kaurenoic acid (KA), a kaurane diterpene found in several medicinal plants, is an active ingredient with potential anti-inflammatory, anticonvulsant, antibacterial and antitumor activities. In this work, an ultra-performance liquid chromatography-tandem mass spectrometry method (UPLC-MS/MS) was firstly developed and validated to quantify kaurenoic acid in rat plasma. Rhein was chosen as the internal standard (IS) and the plasma was processed with one-step acetonitrile protein precipitation; the chromatographic separation was achieved on a HSS T3 (2.1 × 50 mm, 1.8 μm) column with the mobile phase consisting of acetonitrile and water containing 0.1% formic acid via gradient elution. An electrospray ionization source was applied and operated in the negative ion and multiple reaction monitoring (MRM) modes. Kaurenoic acid and IS were quantified using the transitions of m/z 301.2→301.2 (pseudo MRM) and m/z 283.2 → 238.9, respectively. The calibration curves were linear over the range of 5∼ 100 ng/mL (R2 = 0.990). The lower limit of quantification (LLOQ) was 5 ng/mL. The intra- and inter- day precision (RSD) ranged from 3.0% to 11.4%. The matrix effect and extraction recovery were within acceptable limits. The validated method was successfully applied to the pharmacokinetic study of kaurenoic acid in rats after oral administration at three dosages.

A novel LC-MS/MS method for the quantitative measurement of the acetate content in pharmaceutical peptides

Most pharmaceutical peptides are supplied as acetate salts and the relative amount of acetate to peptide in the final product is one quality criterion required by regulatory agencies to approve the product for clinical use. The objective of the present study was to develop a validated LC-MS/MS method that allows the quantitative determination of the acetate content in pharmaceutical peptide preparations and simultaneous monitoring and collection of qualitative and quantitative information on the peptide during manufacture and in the final product. The method uses reversed phase C18-chromatography to elute the acetate ions under acidic conditions, pH 3, followed by post-column infusion of ammonium hydroxide 0.6M, methanolic solution (30:70) at a rate of 0.5mL/hr. The acetate ions were monitored in negative polarity mass spectrometry by pseudo multiple reaction monitoring (pseudo MRM) against 1, 2- 13C labelled acetate, the internal standard used in the method. The method was linear for acetate concentrations between 0.4 and 25μg/mL with a coefficient of determination (r2) equal to 0.9999. The minimum level of detection and minimum level of quantification were at 0.06μg/mL and 0.18μg/mL respectively. Accuracy of the method was judged by determining the acetate content in a commercial product of the peptide pharmaceutical tetracosactide (TCS) and parallel comparison to the amounts determined by a reversed phase HPLC method with detection at a wavelength of 210nm. The amounts determined by the two methods were in agreement with a RSD that was less than 2%. Additional confirmation of method accuracy was determined by spiking the pharmaceutical peptide with varying amounts of acetate. The recoveries ranged on average between 101 and 102% for the spiked amounts. Accuracy was also determined by calculating the percentage relative error of the predicted to actual acetate concentration in quality controls and was determined to be less than 5%. The LC-MS/MS method was precise with an intra- and inter-day RSD of less than 5%. The standard solutions were stable for at least one month when kept frozen at −80°C with no loss in response and an inter-day RSD of less than 5%. The method was applied to quantify the acetate content in the clinically available product of TCS and to simultaneously evaluate the average peptide molecular weight and detect known impurities by switching from negative polarity MRM analysis to positive polarity MS analysis following the elution of the acetate peak. The method reported herein should corroborate quantitative determinations of the acetate content in pharmaceuticals by the traditional compendial HPLC method.

Challenges for Detecting Valproic Acid in a Nontargeted Urine Drug Screening Method.

Valproic acid (VPA) is a widely prescribed medicine, and acute toxicity is possible. As such, it should be included in any nontargeted urine drug screening method. In many published liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS/MS) methods, VPA is usually measured using a pseudo-multiple reaction monitoring (MRM) transition. We investigate a simple ultra-high-performance liquid chromatography-quadrupole time-of-flight (QTof) approach to detect the presence of VPA with more confidence. Three commercially sourced VPA metabolites were characterized and added to a nontargeted high-resolution MS urine drug screening method. All analyses were performed on a Waters Xevo G2-XS LC-QTof in negative electrospray ionization mode. The mass detector was operated in MS mode, and data were processed with UNIFI software. Sixty-eight patient urine samples, which were previously identified by a well-established gas chromatography-MS method as containing VPA, were analyzed on the Waters Xevo G2-XS LC-QTof, to validate this approach. VPA metabolite standards were characterized, and their detection data were added to the broad drug screening library. VPA metabolites were readily detectable in the urine of patients taking VPA. The inclusion of characterized VPA metabolites provides a simple and reliable method enabling the detection of VPA in nontargeted urine drug screening.

Determination of Menadione by Liquid Chromatography-Tandem Mass Spectrometry Using Pseudo Multiple Reaction Monitoring.

This study aimed to develop a menadione (MD) determination method employing liquid chromatography-tandem mass spectrometry (LC-MS/MS) using a pseudo multiple reaction monitoring (MRM) technique, wherein two quadrupoles are used to monitor the same ion. Detection limits of 40 and 2 pg were obtained for MD and its deuterium-labeled form, respectively, whereas MD intra- and inter-assay coefficient of variation values were determined as 5.4 - 8.2%, with the corresponding recoveries equaling 90.5 - 109.6%. The developed method enables determination of MD in urine, plasma, cell extract, and culture media, demonstrating that pseudo multiple reaction monitoring can achieve quantification of compounds forming no suitable product ions, such as MD.

Development and characterization of a pseudo multiple reaction monitoring method for the quantification of human uromodulin in urine.

Uromodulin is the most abundant protein in healthy human urine. Recently it has been suggested as a specific biomarker of renal tubular damage. We have developed a novel pseudo multiple reaction monitoring (pseudo MRM) for the protein's quantification in human urine. Selection of two peptides allowed quantification of uromodulin in human urine. The pseudo MRM quantified uromodulin in healthy individuals between 21 and 1344 nM and in autosomal dominant tubulointerstitial kidney disease-UMOD patients between 2 and 25 nM. The pseudo MRM allows greater confidence in assay specificity than traditional MRM methods and quantified uromodulin at concentrations higher than achievable by ELISA. Differences in urinary uromodulin concentration related to the rs4293393 promoter variant in the UMOD gene was confirmed. This method will be used to further investigate uromodulin as a biomarker of renal injury.

Quantitative analysis of low-abundance serological proteins with peptide affinity-based enrichment and pseudo-multiple reaction monitoring by hybrid quadrupole time-of-flight mass spectrometry

Multiple reaction monitoring (MRM) is commonly used for the quantitative analysis of proteins during mass pectrometry (MS), and has excellent specificity and sensitivity for an analyte in a complex sample. In this study, a pseudo-MRM method for the quantitative analysis of low-abundance serological proteins was developed using hybrid quadrupole time-of-flight (hybrid Q-TOF) MS and peptide affinity-based enrichment. First, a pseudo-MRM-based analysis using hybrid Q-TOF MS was performed for synthetic peptides selected as targets and spiked into tryptic digests of human serum. By integrating multiple transition signals corresponding to fragment ions in the full scan MS/MS spectrum of a precursor ion of the target peptide, a pseudo-MRM MS analysis of the target peptide showed an increased signal-to-noise (S/N) ratio and sensitivity, as well as an improved reproducibility. The pseudo-MRM method was then used for the quantitative analysis of the tryptic peptides of two low-abundance serological proteins, tissue inhibitor of metalloproteinase 1 (TIMP1) and tissue-type protein tyrosine phosphatase kappa (PTPκ), which were prepared with peptide affinity-based enrichment from human serum. Finally, this method was used to detect femtomolar amounts of target peptides derived from TIMP1 and PTPκ, with good coefficients of variation (CV 2.7% and 9.8%, respectively), using a few microliters of human serum from colorectal cancer patients. The results suggest that pseudo-MRM using hybrid Q-TOF MS, combined with peptide affinity-based enrichment, could become a promising alternative for the quantitative analysis of low-abundance target proteins of interest in complex serum samples that avoids protein depletion.

Targeted quantitative phosphoproteomic analysis of erythrocyte membranes during blood bank storage.

One of the hallmarks of blood bank stored red blood cells (RBCs) is the irreversible transition from a discoid to a spherocyte-like morphology with membrane perturbation and cytoskeleton disorders. Therefore, identification of the storage-associated modifications in the protein-protein interactions between the cytoskeleton and the lipid bilayer may contribute to enlighten the molecular mechanisms involved in the alterations of mechanical properties of stored RBCs. Here we report the results obtained analyzing RBCs after 0, 21 and 35 days of storage under standard blood banking conditions by label free mass spectrometry (MS)-based experiments. We could quantitatively measure changes in the phosphorylation level of crucial phosphopeptides belonging to β-spectrin, ankyrin-1, α-adducin, dematin, glycophorin A and glycophorin C proteins. Data have been validated by both western blotting and pseudo-Multiple Reaction Monitoring (MRM). Although each phosphopeptide showed a distinctive trend, a sharp increase in the phosphorylation level during the storage duration was observed. Phosphopeptide mapping and structural modeling analysis indicated that the phosphorylated residues localize in protein functional domains fundamental for the maintenance of membrane structural integrity. Along with previous morphological evidence acquired by electron microscopy, our results seem to indicate that 21-day storage may represent a key point for the molecular processes leading to the erythrocyte deformability reduction observed during blood storage. These findings could therefore be helpful in understanding and preventing the morphology-linked mechanisms responsible for the post-transfusion survival of preserved RBCs.

Pharmacokinetic study of HS061, a new human insulin, in non-diabetic rat using ultra performance liquid chromatography–tandem mass spectrometry

HS061, a new structure analogue of human insulin, was investigated for the treatment of diabetes. In this study, we developed a simple and accurate UPLC-MS/MS method for the pharmacokinetic studies of HS061 in non-diabetic rats followed by a full method validation. Following a simple protein precipitation with acetonitrile, the analyte and internal standard (Levemir, IS) were separated on a Waters XBridge™ BEH300 C4 column (100 mm × 4.6 mm i.d., 3.5 μm) with a gradient elution using acetonitrile and 0.2% aqueous formic acid. The method was operated under pseudo-multiple reaction monitoring (pseudo-MRM) in the positive electrospray ionization mode. The monitored transitions were set at m/z 1563.4→1563.4 for HS061 by pseudo-MRM and m/z 1184.7→454.5 for IS by MRM. Linear calibration curves were obtained over the concentration ranges of 10-1000 ng/mL and no interfering peaks were detected at the retention time of HS061 and IS in blank rat plasma. The mean extraction recoveries of HS061 at three concentrations of 20, 100, 800 ng/mL were greater than 95.17%. Stability was assessed under different conditions and no significant degradations were found. The validated method was then successfully applied in measuring HS061 following subcutaneous (0.5, 1.0, 3.0 U/kg) and intravenous (1.0 U/kg) injection in rat plasma to support the pre-clinical pharmacokinetic study. Maximum plasma concentration (Cmax) and area under the curve (AUC) for the subcutaneous doses of HS061 was approximately dose proportional while other pharmacokinetic parameters showed no significant differences among the three doses (p>0.05). The absolute bioavailability of HS061 after subcutaneous administration at 1.0 U/kg was estimated to be 70.40%.

Rapid and sensitive method for the determination of polycyclic aromatic hydrocarbons in soils using pseudo multiple reaction monitoring gas chromatography/tandem mass spectrometry

A method for the rapid determination of 18 polycyclic aromatic hydrocarbons (PAHs) in soil has been established based on a simplified solvent extraction and GC/MS/MS operated in pseudo multiple reaction monitoring mode (PMRM), a technique where the two quadrupoles mass monitor the same m/z. The PMRM approach proved superior to the classic single quadrupole technique, with enhanced sensitivity, specificity, and significant reduction in time consuming sample clean-up procedures. Trace level PAHs could be readily confirmed by their retention times and characteristic ions. The limit of quantitation in soil was observed to be 20ng/g for 16 EPA-priority PAHs and 2 additional PAHs specific to Environment Canada. The developed method was linear over the calibration range 20–4000ng/g in soil, with observed coefficients of determination of >0.996. Individual PAH recoveries from fortified soil were in the range 58.1 to 110.1%, with a precision between 0.3 and 4.9% RSD. The ruggedness of the method was demonstrated by the success of an inter-lab proficiency test study organized by the Canadian Association for Laboratory Accreditation. The present method was found to be applicable as a rapid, routine screening for PAH contamination in soil, with significant savings in terms of preparation time and solvent usage.

A method for the analysis of sucralose with electrospray LC/MS in recipient waters and in sewage effluent subjected to tertiary treatment technologies

A method for the analysis of the artificial sweetener sucralose in sewage water and recipient water was developed. Extraction and clean up was performed with solid-phase extraction utilising Oasis HLB columns. Detection was made by liquid chromatography electrospray mass spectrometry (LC/MS). The triple-quadrupole mass spectrometer was operated in multiple reaction monitoring (MRM) mode. However, ‘pseudo MRM’ was used, a technique where the two quadrupoles monitor the same m/z. The sodium adduct of sucralose was used for quantification, since lower detection limits were obtained as compared to the sucralose quasi-molecular ion in negative ion mode. The two ions with highest intensity in the chlorine isotope pattern were monitored. The reduction of matrix effects with this approach is discussed. The method limit of quantification (MLOQ) for sewage water was 0.2 µg L−1, whereas for recipient water MLOQ was 0.02 µg L−1. The method was used to analyse effluent samples from an experimental sewage treatment plant (STP) to assess the efficiency of tertiary treatment techniques for removal of sucralose. Filtration through activated carbon was shown to be efficient, while ozonation, advanced oxidation techniques and membrane bioreactors were less efficient. Analyses of receiving waters showed low dilution of sucralose emitted from the STPs.

Use of a Solvent-Free Dry Matrix Coating for Quantitative Matrix-Assisted Laser Desorption Ionization Imaging of 4-Bromophenyl-1,4-diazabicyclo(3.2.2)nonane-4-carboxylate in Rat Brain and Quantitative Analysis of the Drug from Laser Microdissected Tissue Regions

A dry matrix application for matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) was used to profile the distribution of 4-bromophenyl-1,4-diazabicyclo(3.2.2)nonane-4-carboxylate, monohydrochloride (BDNC, SSR180711) in rat brain tissue sections. Matrix application involved applying layers of finely ground dry alpha-cyano-4-hydroxycinnamic acid (CHCA) to the surface of tissue sections thaw mounted onto MALDI targets. It was not possible to detect the drug when applying matrix in a standard aqueous-organic solvent solution. The drug was detected at higher concentrations in specific regions of the brain, particularly the white matter of the cerebellum. Pseudomultiple reaction monitoring imaging was used to validate that the observed distribution was the target compound. The semiquantitative data obtained from signal intensities in the imaging was confirmed by laser microdissection of specific regions of the brain directed by the imaging, followed by hydrophilic interaction chromatography in combination with a quantitative high-resolution mass spectrometry method. This study illustrates that a dry matrix coating is a valuable and complementary matrix application method for analysis of small polar drugs and metabolites that can be used for semiquantitative analysis.