Multiple Reaction Monitoring Experiments Research Articles

Rapid screening procedures for a variety of complex forensic samples using laser diode thermal desorption (LDTD) coupled to different mass spectrometers.

The applications shared in this paper demonstrate the wide variety of samples that can be analyzed when Laser Diode Thermal Desorption (LDTD) is interfaced with a high-resolution mass spectrometer and show the speed at which high quality data can be generated from complex matrices. Samples are solvent extracted and spotted in a 96-well plate. In the case of biological fluids, hydrolysis followed by solid-phase extraction is required. The solvent in the 96-well plate is evaporated followed by mass spectrometric (MS) analysis with atmospheric pressure chemical ionization. Where applicable, the instrument is operated in data-dependent mode, with a full-scan mass spectrum followed by MS/MS spectra of the top 10 ions with a total runtime of 0.4 min. Four applications (MAAQ and Tear Gas, twelve rodenticides, seven explosives, and 40 drugs of abuse) are reported in this paper. MAAQ, tear gas, and rodenticides were identified by full-scan, followed by MS/MS experiments at levels of 125 μg/L, 125 μg/L, and 500 μg/L, respectively. Explosives were all identified at 102 μg/L by full-scan experiments. The drugs of abuse were identified by multiple reaction monitoring (MRM) experiments at defined cutoff levels from 2 to 1000 μg/L. Interfacing LDTD with a mass spectrometer allows for rapid screening of a wide range of samples, with either minimal or complex sample preparation. Using a high-resolution mass spectrometer with the combination to perform full-scan and MS/MS experiments adds a high level of specificity.

Corylus avellana: A Source of Diarylheptanoids With α-Glucosidase Inhibitory Activity Evaluated by in vitro and in silico Studies.

Corylus avellana hard shells, green leafy involucres, leaves, and male flowers have shown to be a source of diarylheptanoids, a class of natural products with promising biological activities. Cyclic diarylheptanoids, named giffonins, were isolated from the Italian cultivar “Tonda di Giffoni.” Even if many efforts have been made to establish the chemistry of these compounds, little is known about their biological properties. Herein, the inhibitory effects of diarylheptanoids isolated from C. avellana byproducts against α-glucosidase enzyme were evaluated. Molecular docking experiments disclosed the establishment of several key interactions between all the screened diarylheptanoids and the protein counterpart, whose model was built through homology modeling procedure, thus rationalizing the detected inhibitory activities. Specifically, the most active compounds giffonin J (10), K (11), and P (16) were able to make both H-bonds and π–π stacking contacts with different residues belonging to the binding site responsible for the catalytic activity of the investigated enzyme. To highlight the occurrence of the bioactive diarylheptanoids in the extracts of C. avellana byproducts obtained by eco-friendly extractions, their LC-MS profiles were analyzed. LC-MS analysis showed how giffonin J (10), K (11), and P (16) occurred in the ethanol extract of the leaves, while in the extracts of shells and green leafy involucres only giffonin P (16) was evident. Moreover, the quantitative analysis of giffonin J (10), K (11), and P (16) in C. avellana byproducts was carried out by an analytical approach based on LC–ESI/QTrap/MS, using the Multiple Reaction Monitoring (MRM) experiment. These results prompt to evaluate C. avellana byproducts, especially the leaves, as a prospective source of bioactive diarylheptanoids for the development of functional ingredients for the treatment of diabetes.

Validation of Analytical Methods for Tacrolimus Determination in Poly(ε-caprolactone) Nanocapsules and Identification of Drug Degradation Products.

The aim of this paper was to use chromatographic tools for validating an analytical method for the tacrolimus (TAC) determination in polymeric nanocapsules and for identifying the drug degradation products after alkaline stress. A rapid Ultra-High-Performance Liquid Chromatography coupled with photo-diode array (UHPLC-PDA) method was successfully performed using the following chromatographic conditions: the Shimadzu Shim-pack XR-ODS III C18 column (100 mm×2.00 mm, 2.2 μm), the mobile phase consisting of methanol and acidified ultrapure water (89:11 v/v), the flow rate of 0.55 mL·min-1, and the ultraviolet (UV) detection at 235 nm. This method was validated as per International Council for Harmonisation (ICH) guidelines. In addition, a TAC forced degradation assay was carried out after alkaline stress and its degradation products were investigated using Liquid Chromatography coupled tandem mass spectroscopy (LC-MS/MS). The calibration curve was linear in the range of 100.0-300.0 μg·mL-1 (r >0.9999). Accuracy was confirmed by the TAC recovery of 96.55 to 98.19%. Precision (intraday and interday) were demonstrated by relative standard deviation lower than 0.89% and 3.25%, respectively. Selectivity and robustness were also proved. The method developed it was successfully applied to quantify TAC from polymeric nanocapsules, showing a high loading efficiency rate (>96.47%). The main drug degradation product observed in a multiple reaction monitoring (MRM) experiment was m/z 844, confirming the susceptibility of TAC under alkaline conditions; this finding was first time described.

Quantitative Analysis of Bioactive Carbazole Alkaloids in Murraya koenigii (L.) from Six Different Climatic Zones of India Using UPLC/MS/MS and Their Principal Component Analysis.

Murraya koenigii (L.) Spreng (Curry leaf) is a commercially important medicinal plant in South Asia, containing therapeutically valuable carbazole alkaloids (CAs). Thus, the quantitative evaluation of these compounds from different climatic zones of India are an important aspect for quality assessment and economic isolation of targeted compounds from the plant. In this study, quantitative estimation of CAs among 34 Indian natural populations of M. koenigii was assessed using UPLC/MS/MS. The collected populations represent the humid subtropical, tropical wet & dry, tropical wet, semi-arid, arid, and montane climatic zones of India. A total of 11 CAs viz. koenine-I, murrayamine A, koenigine, koenimbidine, koenimbine, O-methylmurrayamine A, girinimbine, mahanine, 8,8''-biskoenigine, isomahanimbine, and mahanimbine were quantified using multiple reaction monitoring (MRM) experiments within 5.0 min. The respective range for natural abundance of CAs were observed as 0.097-1.222, 0.092-5.014, 0.034-0.661, 0.010-1.673, 0.013-7.336, 0.010-0.310, 0.010-0.114, 0.049-5.288, 0.031-1.731, 0.491-3.791, and 0.492-5.399 mg/g in leaves of M. koenigii. The developed method shown linearity regression coefficient (r2 >0.9995), LOD (0.003-0.248 ng/mL), LOQ (0.009-0.754 ng/mL), and the recovery was between 88.803-103.729 %. The bulk of these CAs were recorded in their highest concentrations in the humid subtropical zone, followed by the tropical wet & dry zones of India. Further, principal component analysis (PCA) was performed which differentiated the climatic zones according to the dominant and significant CAs contents within the populations. The study concludes that the method established is simple, rapid, with high sample throughput, and can be used as a tool for commercial purposes and quality control of M. koenigii.

Protein Targets of Acetaminophen Covalent Binding in Rat and Mouse Liver Studied by LC-MS/MS.

Acetaminophen (APAP) is a mild analgesic and antipyretic used commonly worldwide. Although considered a safe and effective over-the-counter medication, it is also the leading cause of drug-induced acute liver failure. Its hepatotoxicity has been linked to the covalent binding of its reactive metabolite, N-acetyl p-benzoquinone imine (NAPQI), to proteins. The aim of this study was to identify APAP-protein targets in both rat and mouse liver, and to compare the results from both species, using bottom-up proteomics with data-dependent high resolution mass spectrometry and targeted multiple reaction monitoring (MRM) experiments. Livers from rats and mice, treated with APAP, were homogenized and digested by trypsin. Digests were then fractionated by mixed-mode solid-phase extraction prior to liquid chromatography-tandem mass spectrometry (LC-MS/MS). Targeted LC-MRM assays were optimized based on high-resolution MS/MS data from information-dependent acquisition (IDA) using control liver homogenates treated with a custom alkylating reagent yielding an isomeric modification to APAP on cysteine residues, to build a modified peptide database. A list of putative in vivo targets of APAP were screened from data-dependent high-resolution MS/MS analyses of liver digests, previous in vitro studies, as well as selected proteins from the target protein database (TPDB), an online resource compiling previous reports of APAP targets. Multiple protein targets in each species were found, while confirming modification sites. Several proteins were modified in both species, including ATP-citrate synthase, betaine-homocysteine S-methyltransferase 1, cytochrome P450 2C6/29, mitochondrial glutamine amidotransferase-like protein/ES1 protein homolog, glutamine synthetase, microsomal glutathione S-transferase 1, mitochondrial-processing peptidase, methanethiol oxidase, protein/nucleic acid deglycase DJ-1, triosephosphate isomerase and thioredoxin. The targeted method afforded better reproducibility for analysing these low-abundant modified peptides in highly complex samples compared to traditional data-dependent experiments.

Exploratory analysis using MRM profiling mass spectrometry of a candidate metabolomics sample for testing system suitability

Multiple reaction monitoring (MRM) profiling is an exploratory mass spectrometry (MS) method applicable to the initial screening of complex samples for small molecules based on their chemical functionalities. We report on the applicability and quality of this method to screen for metabolites, lipids and exogenous compounds in a candidate reference material, the Metabolomics System Suitability Research Grade Material (RGM 10122) which is being developed by the National Institute of Standards and Technology (NIST). In an initial discovery experiment, we recorded data using eighty neutral loss (NL) and precursor ion (Prec) MS/MS scans, selected from literature data as likely of value in recognizing the presence of potential lipids and metabolites. Then the NIST sample was re-examined combining precursor-to-product ion transitions from the discovery experiment with a list of 1357 known literature-based metabolite MRMs. This MRM profiling experiment gave a small set (191) of high-quality lipid specific MRMs for the sample. Similar experiments gave 104 and 17 metabolite and exosome MRM's. These MRM experiments, with a few exceptions, showed relative standard deviations (RSD) under 25% for individual tentatively assigned compounds. At a data acquisition rate of 50 compounds/min, using two MRMs for each compound, this approach allows quick surveys for easily detectable compounds in complex samples.

Determination of individual oligonucleotide impurities by small amine ion pair-RP HPLC MS and MS/MS: n − 1 impurities

Ion pair-reversed phase (IP-RP) HPLC is one of the most widely used methods for the analysis of oligonucleotide impurities. The method is compatible with mass spectrometry and has been used to guide the development of improved synthesis and purification approaches. The ability to detect and characterize impurities depends on the reagents and the IP buffer system employed, as each can directly affect the degree of chromatographic separation and the sensitivity of detection by MS. Previous work in our laboratory has shown that small alkyl amines are suitable IP reagents for the analysis of impurities in phosphate diester oligonucleotides and can be used to differentiate among individual members of composite impurity families. The addition of an alkyl acid often further enhances peak separation, but at the detriment of ion signal. An improved method with increased chromatographic performance and sensitivity of detection is presented here. Improvements were mainly realized through the use of lower concentrations of small alkyl amine (i.e., 5 mM) and acid (0.5 mM) IP reagents, and ammonium bicarbonate (20 mM) as a buffer. The improved capabilities of the new method are demonstrated by separation of the individual components of the composite n − 1 impurity in a set of four production-scale batches of a single oligonucleotide. Addition of the alkyl acid resulted in resolution of most individual n − 1 impurities. The observed enhanced sensitivity of detection allowed multiple reaction monitoring (MRM) experiments, which were used to differentiate among unresolved impurities.

Solid phase extraction and LC-MS/MS quantification of ibandronate in human plasma

Purpose: To develop and validate a simple, highly sensitive and accurate method for the quantification of ibandronate (IBN) in human plasma.Methods: Electrospray ionization liquid chromatography tandem mass spectrometry (LC-MS/MS) in positive ion mode was applied to obtain optimum signals. The parent ion was acquired under collisionactivated dissociation conditions, and the abundant fragments used to design multiple reaction monitoring experiments for monitoring two ibandronate transitions (m/z 376 to m/z 114, and m/z 376 to m/z 250). The IBN was isolated from plasma with weak anion exchange solid phase extraction columns with ‘on-cartridge’ derivatization using tri-methylsilyl-diazomethane (TMSDZ) reagent to convert IBN to tetra-methyl derivative.Results: The studied drug was successfully extracted from plasma samples without any interference at a retention time of 3.2 min. The matrix effect averaged 110 %, indicating that endogenous materials had little effect on ionization. The relationship between plasma analyte concentration and IBN signal area was satisfactorily linear, with correlation coefficient (r2) ranging from 0.9817 to 0.9942 in the concentration range of 0.5 – 200 ng/ml. The lower and upper limits of quantification (LLOQ and ULOQ) for IBN were 0.5 and 200 ng/ml, respectively. Relative recovery of IBN from plasma after extraction and derivatization at 3 distinct concentrations was 83.93 to 85.06 %, relative to standard solutions. The ranges of intra- and inter-day accuracies of quantification of quality controls were 89.39 - 106.40 %, and 90.50 - 107.96 %, respectively. Processed plasma IBN extracts were stable in autosampler at 4 0C (91.12 to 103.49%). Long-term stability in plasma after 30 days at -24 0C ranged from 89.52 to 113.18 %.Conclusion: This validated LC-MS/MS method can be successfully applied for determination of IBN in pharmacokinetic studies. It is a sensitive and specific assay for plasma IBN in bioequivalence studies.
 Keywords: Ibandronate, LC-MS/MS, Validation, Derivatization, Solid-phase extraction

PB-Net: Automatic peak integration by sequential deep learning for multiple reaction monitoring

Mass spectrometry (MS) based proteomics has become an indispensable component of modern molecular and cellular biochemistry analysis. Multiple reaction monitoring (MRM) is one of the most well-established MS techniques for molecule detection and quantification. Despite its wide usage, there lacks an accurate computational framework to analyze MRM data, and expert annotation is often required, especially to perform peak integration. Here we propose a deep learning method PB-Net (Peak Boundary Neural Network), built upon recent advances in sequential neural networks, for fully automatic chromatographic peak integration. To train PB-Net, we generated a large dataset of over 170,000 expert annotated peaks from MS transitions spanning a wide dynamic range, including both peptides and intact glycopeptides. Our model demonstrated outstanding performances on unseen test samples, reaching near-perfect agreement (Pearson's r 0.997) with human annotated ground truth. Systematic evaluations also show that PB-Net is substantially more robust and accurate compared to previous state-of-the-art peak integration software. PB-Net can benefit the wide community of mass spectrometry data analysis, especially in applications involving high-throughput MS experiments. Codes and test data used in this work are available at https://github.com/miaecle/PB-net. SignificanceHuman annotations serve an important role in accurate quantification of multiple reaction monitoring (MRM) experiments, though they are costly to collect and limit analysis throughput. In this work we proposed and developed a novel technique for the peak-integration step in MRM, based on recent innovations in sequential deep learning models. We collected in total 170,000 expert-annotated MRM peaks and trained a set of accurate and robust neural networks for the task. Results demonstrated a substantial improvement over the current state-of-the-art software for mass spectrometry analysis and comparable level of accuracy and precision as human annotators.

Prediction of LC-MS/MS Properties of Peptides from Sequence by Deep Learning

Deep learning models for prediction of three key LC-MS/MS properties from peptide sequences were developed. The LC-MS/MS properties or behaviors are indexed retention times (iRT), MS1 or survey scan charge state distributions, and sequence ion intensities of HCD spectra. A common core deep supervised learning architecture, bidirectional long-short term memory (LSTM) recurrent neural networks was used to construct the three prediction models. Two featurization schemes were proposed and demonstrated to allow for efficient encoding of modifications. The iRT and charge state distribution models were trained with on order of 105 data points each. An HCD sequence ion prediction model was trained with 2 × 106 experimental spectra. The iRT prediction model and HCD sequence ion prediction model provide improved accuracies over the start-of-the-art models available in literature. The MS1 charge state distribution prediction model offers excellent performance. The prediction models can be used to enhance peptide identification and quantification in data-dependent acquisition and data-independent acquisition (DIA) experiments as well as to assist MRM (multiple reaction monitoring) and PRM (parallel reaction monitoring) experiment design.

Optimization and validation of a liquid chromatography/tandem mass spectrometry (LC-MS/MS) method for the determination of aflatoxins in maize

A LC-MS/MS method has been optimized and validated for the determination of aflatoxins (AFB1, AFB2, AFG1 and AFG2) in maize. Extraction was performed using a modified QuEChERS method with little sample preparation without the need for purification procedure. Determination was performed by high pressure liquid chromatography (HPLC) coupled to tandem mass spectrometry (MS/MS). The acquisition was performed using MassHunter software in Multiple Reaction Monitoring (MRM) mode in positive polarity. Different mobile phases were tested to control the degree of the ionization and good performances were obtained for methanol/water with 5 mM ammonium acetate. MRM experiments were optimized for each aflatoxin in order to generate sensitive transitions. Linearity was demonstrated for the aflatoxins in the range 0.225–1.25 μg/L. Limits of detection (LOD) (0.11 and 0.36 μg/Kg) and limits of quantification (LOQ) (0.36–1.19 μg/Kg) of the aflatoxins are below the maximum permitted levels set by the European Union (EU). Aflatoxins have acceptable recoveries using QuEChERS method in the acceptable range of 50–120% for levels below 1 μg/Kg. Satisfactory recoveries were also obtained in the acceptable range of 70–110% for levels between 1 and 10 μg/Kg except for AFB2. Relative standard deviation (RSD) of recoveries for the intra-day precision and inter-day precision were below 11 %. Selectivity of the method was tested and no spectral interferences were observed in the appropriate retention times. The main advantage of the proposed method is its ease of use and requires a smaller solvent consumption that reduces the time and cost of the analysis.

The study and application of analyte adduct based ionisation of propofol in the analysis with liquid chromatography-tandem mass spectrometry

The ionisation of propofol was studied on the basis of analyte adduct formation with the components of the mobile phase. The identification of an appropriate propofol adduct ion generated in an acetate salt based buffer in the negative electrospray ionisation mode made the propofol LC-MS/MS analysis with two multiple reaction monitoring experiments possible without analyte derivatisation. Since a product ion of the deprotonated analyte was also used in the detection, these two fragmentation experiments were performed with different precursor ions. Liquid-liquid extraction with 1-chlorobutane was applied for sample preparation. A Luna 5 μm C18 (2) 100 A, 150 mm × 2 mm analytical column together with a mobile phase composed of water/methanol (3/97, v/v) with 10 mmol·L−1 ammonium acetate and 0.1% acetic acid were applied. The limit of detection and quantification were 0.02 mg/L and 0.06 mg/L appropriate. Other results of the validation experiments performed like linearity of the calibration range, accuracy and precision, processed sample stability, recovery, matrix effect and selectivity revealed that this propofol quantification strategy developed can be defined as an applicable alternative to other methods presented.

Quantification of 1,3-olein-2-palmitin (OPO) and Palmitic Acid in sn-2 Position of Triacylglycerols in Human Milk by Liquid Chromatography Coupled with Mass Spectrometry.

This study describes the identification and quantification of fatty acids in the sn-2 position of triacylglycerols (TAG) and of the most abundant TAG regioisomers in human milk by liquid chromatography coupled with high-resolution mass spectrometry (HPLC-HRMS). Over 300 individual TAG species were observed and 1,3-olein-2-palmitin (OPO) was identified as the most abundant TAG regioisomer. Validation of the HPLC-HRMS method showed repeatability and intermediate reproducibility values ranging from 3.1 to 16.6% and 4.0 to 20.7%, respectively, and accuracy ranging from 75 to 97%. Results obtained by the HPLC-HRMS method were comparable to results from the ISO 6800 method for the quantification of palmitic acid in the sn-2 position of TAG (81.4 and 81.8 g 100 g−1 total palmitic acid, respectively). Processing the data obtained with the HPLC-HRMS method is extremely time consuming and, therefore, a targeted method suitable for the quantification of OPO in human milk samples by ultra-performance (UP) LC coupled with triple quadrupole (QQQ) MS was developed and validated. OPO identification and quantification by UPLC-QQQ were based on nominal mass and a fragmentation pattern obtained by multiple reaction monitoring experiments. The method was validated in terms of accuracy and precision by analyzing different aliquots of the same human milk sample over time and comparing the results with values obtained by HPLC-HRMS. Intermediate reproducibility was <15% and trueness comparable to HPLC-HRMS. Quantification of OPO in human milk samples collected at 30, 60 and 120 days postpartum showed that OPO content varies between 333 ± 11.8 and 383 ± 18.0 mg 100mL−1.

Comprehensive identification and quantification of chlorogenic acids in sweet cherry by tandem mass spectrometry techniques

This paper deals with an extensive analytical approach, which uses two complementary tandem mass spectrometry techniques, to characterize the chlorogenic acids (CGAs) present in a typical Italian sweet cherry variety (cv Ferrovia). Sixteen monoacyl-quinic acids and esters, five diacyl-quinic acids, three caffeoyl-quinic acids glycosides, and two caffeic acid hexoses were detected by HPLC-MSn analyses (MSn up to MS4), among which four methyl coumaroyl quinate and three methyl caffeoyl quinate isomers were tentatively identified in sweet cherries for the first time. HPLC-MS/MS analyses through multiple reaction monitoring (MRM) experiments showed that trans-3-O-caffeoylquinic acid, cis-3-O-coumaroylquinic acid, trans-3-O-coumaroylquinic acid, trans-5-O-caffeoylquinic acid, and methyl-3-O-caffeoyl quinate were the main CGAs in the mature berries of cv Ferrovia. Considering that CGAs can have several health benefits depending on their amount but also on their structural features, the results of this study provide new insight into the knowledge of the quali-quantitative profile of these phytochemicals in a widespread fruit such as sweet cherry.

Adduct Formation-Supported Two-Way Electrospray Ionization Strategy for the Determination of Urinary Creatinine Concentration with LC-MS-MS in Abstinence Control.

In this work, an alternative LC-MS-MS strategy for the analysis of urinary creatinine in abstinence control was presented and discussed. The two-way electrospray ionization consisted of two different precursor ions in which fragmentation was used in multiple reaction monitoring experiments. A creatinine adduct ion with sodium and sodium acetate together with the conventional analyte protonation was investigated. Adduct formation and fragmentation was explored by appropriate infusion experiments performed with analyte solutions prepared in different concentrations. The analytical signal was compensated by the application of appropriate isotopically labeled internal standard. The advantages of information carried by precursor ions separated in the mass spectra were pointed out. Sample preparation based solely on sample dilution performed in the final HPLC vial directly. A Luna 5 μm C18 (2) 100 A, 150 mm × 2 mm analytical column together with a mobile phase consisted of H2O/methanol = 3%/97% (v/v) with 10 mmol/L ammonium acetate and 0.1% acetic acid (flow = 0.4 mL/min) were used for the separation performed during a run of 5 min. The linearity was examined in the range of 100-3,000 mg/L. The limit of detection (13 mg/L), limit of quantification (43 mg/L) together with method precision/accuracy, selectivity, stability and matrix effect were tested to be appropriate for forensic applications. The applicability of water as surrogate matrix for method calibration was also examined successfully. The presented strategy was used in the analysis of real samples. No interferences with the creatinine peak eluted at about 1.0 min could be recorded.

Carotenoids and apocarotenoids determination in intact human blood samples by online supercritical fluid extraction-supercritical fluid chromatography-tandem mass spectrometry

A direct on-line method based on the coupling of supercritical fluid extraction and supercritical fluid chromatography with triple quadrupole mass spectrometry detection (SFE-SFC-QqQ/MS) for selected carotenoids determination and apocarotenoids detection in intact human blood was developed for the first time. Carotenoids and apocarotenoids were identified by using the available standard together with full scan, selected ion monitoring (SIM), and multiple reaction monitoring (MRM) experiments. Moreover, β-Cryptoxanthin, Zeaxanthin, β-Carotene and Capsanthin were directly quantified by the developed methodology, using a multiple reaction monitoring (MRM) approach; the determined average content of β-carotene was 123.8 nmol L−1 (range 18.7–485.1 nmol L−1), of β-cryptoxanthin was 385.3 nmol L−1 (range 72.5–1920.3 nmol L−1), of zeaxanthin was 396.9 nmol L−1 (range < LoD – 1795.8 nmol L−1) and of capsanthin was 38.9 nmol L−1 (range < LoD – 188.4 nmol L−1). Analyses were carried out on 10 μL aliquots of intact blood samples without any preliminary treatment; the online extraction and chromatographic separation time was just over 20 min. The method was validated in terms of linearity, precision, limits of detection and quantification, and accuracy. Interestingly, β-apo-12′-carotenal, apo-10′-zeaxanthinal, apo-12′-zeaxanthinal, apo-14′-zeaxanthinal, ε-apo-8-luteinal, ε-apo-12-luteinal and ε-apo-14-luteinal were detected in human blood, together with two zeaxanthin fatty acid esters, for the first time.

Microfluidic Separation Coupled to Mass Spectrometry for Quantification of Peanut Allergens in a Complex Food Matrix.

Peanut is an important food allergen, but it cannot currently be reliably detected and quantified in processed foods at low levels. A level of 3 mg protein/kg is increasingly being used as a reference dose above which precautionary allergen labeling is applied to food products. Two exemplar matrices (chocolate dessert and chocolate bar) were prepared and incurred with 0, 3, 10, or 50 mg/kg peanut protein using a commercially available lightly roasted peanut flour ingredient. After simple buffer extraction employing an acid-labile detergent, multiple reaction monitoring (MRM) experiments were used to assess matrix effects on the detection of a set of seven peptide targets derived from peanut allergens using either conventional or microfluidic chromatographic separation prior to mass spectrometry. Microfluidic separation provided greater sensitivity and increased ionization efficiency at low levels. Individual monitored transitions were detected in consistent ratios across the dilution series, independent of matrix. The peanut protein content of each sample was then determined using ELISA and the optimized MRM method. Although other peptide targets were detected with three transitions at the 50 mg/kg peanut protein level in both matrices, only Arah2(Q6PSU2)147-155 could be quantified reliably and only in the chocolate dessert at 10 mg/kg peanut protein. Recoveries were consistent with ELISA analysis returning around 30-50% of the incurred dose. MS coupled with microfluidic separation shows great promise as a complementary analytical tool for allergen detection and quantification in complex foods using a simple extraction methodology.

Determination of the bioavailability of selected pharmaceutical residues in fish plasma using liquid chromatography coupled to tandem mass spectrometry

Aquatic systems near major urban centers are constantly contaminated with effluent from wastewater treatment plants. Pharmaceuticals are part of the contamination and several classes of drugs have been detected in surface waters in the last decade. To better understand the impact of those pharmaceuticals in ecosystems, the exposure to aquatic species needs to be investigated. This study presents a new simple and rugged quantitative method for the determination of several classes of drugs using 100μL of plasma from fish environmentally exposed to a major but highly diluted urban effluent. Six common drugs (i.e., diclofenac, ibuprofen, naproxen, salbutamol, sulfamethoxazole and trimethoprim) and one major metabolite (2-hydroxy-ibuprofen), present in significant amount in impacted waterways have been selected for the development and validation of the method. First, all drugs were extracted using cation exchange solid phase extraction (SPE) and eluted with two solvent mixtures. Then, the extracts were analyzed using a reverse-phase analytical column Waters® CORTECS C18+ (150×2.1mm, 2.7μm) within 14min. MS/MS was performed with an electrospray (ESI) interface in positive ion mode, with multiple reaction monitoring (MRM) experiment acquiring two product ions per drugs. Quantification has been made with standard curves for each analyte using isotopically labeled internal standards. This method has high sensitivity with limits of quantification of 1ngmL−1 for each drug, except for ibuprofen and its metabolite 2-hydroxy-ibuprofen at 2ngmL−1. The precision of the method was below 11%, the accuracy between 94 and 105% and overall recovery between 94 and 111% for all drugs, with high selectivity. Application of the method to plasma samples from wild northern pike inhabiting the St. Lawrence River collected over a three-year period showed the presence of naproxen, diclofenac, trimethoprim and salbutamol at very low concentrations (around 1ngmL−1).

Optimization of Sample Preparation and Instrumental Parameters for the Rapid Analysis of Drugs of Abuse in Hair samples by MALDI-MS/MS Imaging

Matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) has been employed to rapidly screen longitudinally sectioned drug user hair samples for cocaine and its metabolites using continuous raster imaging. Optimization of the spatial resolution and raster speed were performed on intact cocaine contaminated hair samples. The optimized settings (100 × 150 μm at 0.24 mm/s) were subsequently used to examine longitudinally sectioned drug user hair samples. The MALDI-MS/MS images showed the distribution of the most abundant cocaine product ion at m/z 182. Using the optimized settings, multiple hair samples obtained from two users were analyzed in approximately 3 h: six times faster than the standard spot-to-spot acquisition method. Quantitation was achieved using longitudinally sectioned control hair samples sprayed with a cocaine dilution series. A multiple reaction monitoring (MRM) experiment was also performed using the ‘dynamic pixel’ imaging method to screen for cocaine and a range of its metabolites, in order to differentiate between contaminated hairs and drug users. Cocaine, benzoylecgonine, and cocaethylene were detectable, in agreement with analyses carried out using the standard LC-MS/MS method.Graphical ᅟ

Quantitative Mass Spectrometry by Isotope Dilution and Multiple Reaction Monitoring (MRM).

Selected reaction monitoring (SRM) is used in molecular profiling to detect and quantify specific known proteins in complex mixtures. Using isotope dilution (Barnidge et al., Anal Chem 75(3):445-451, 2003) methodologies, peptides can be quantified without the need for an antibody-based method. Selected reaction monitoring assays employ electrospray ionization mass spectrometry (ESI-MS) followed by two stages of mass selection: a first stage where the mass of the peptide ion is selected and, after fragmentation by collision-induced dissociation (CID), a second stage (tandem MS) where either a single (e.g., SRM) or multiple (multiple reaction monitoring, MRM) specific peptide fragment ions are transmitted for detection. The MRM experiment is accomplished by specifying the parent masses of the selected endogenous and isotope-labeled peptides for MS/MS fragmentation and then monitoring fragment ions of interest, using their intensities/abundances and relative ratios to quantify the parent protein of interest. In this example protocol, we will utilize isotope dilution MRM-MS to quantify in absolute terms the total levels of the protein of interest, ataxia telangiectasia mutated (ATM) serine/threonine protein kinase. Ataxia telangiectasia mutated (ATM) phosphorylates several key proteins that initiate activation of the DNA damage checkpoint leading to cell cycle arrest.