Structural characterization of intact antibodies by high‐resolution LTQ Orbitrap mass spectrometry

Recombinant monoclonal antibodies (MAbs) can be heterogeneous due to modifications that can occur during expression, purification or during storage. These large multichain proteins (～150 kDa) are structurally challenging for detailed characterization to identify the sites of modifications. We report the use of LTQ Orbitrap mass spectrometry to accurately measure the average masses of individual glycoforms by direct infusion of an intact antibody. To identify the site‐specific modification of methionines in the antibody caused by forced oxidation, we used a ‘middle‐down’ approach. The antibody was subjected to limited digestion using the endoproteinase Lys‐C and reduced to generate Fab heavy chain, single chain Fc and light chain fragments (～25 kDa each). These species were subjected to on‐line liquid chromatography/mass spectrometry/mass spectrometry (LC/MS/MS) analysis using an LTQ Orbitrap, where these large precursors were dissociated by higher‐energy collisions in the C‐trap. High resolution and accuracy achieved for resulting fragments allowed us to show in a site‐specific manner that only the methionines in the Fc heavy chain were oxidized under the studied conditions. Copyright © 2009 John Wiley & Sons, Ltd.     

Characterization of xylazine metabolism in rat liver microsomes using liquid chromatography–hybrid triple quadrupole–linear ion trap–mass spectrometry

ABSTRACT: Xylazine is an α₂‐adrenoceptor agonist and it is widely used in veterinary anesthesia in combination with ketamine. There is limited information on the metabolism of xylazine. A quantitative method for the determination of xylazine by HPLC‐ESI/MS/MS was developed. The method consisted of a protein precipitation extraction followed by analysis using liquid chromatography electrospray tandem mass spectrometry. The chromatographic separation was achieved using a Thermo Betasil Phenyl 100 × 2 mm column combined with an isocratic mobile phase composed of acetonitrile, methanol, water and formic acid (60:20:20:0.4) at a flow rate of 300 μL/min. The mass spectrometer was operating in selected reaction monitoring mode and the analytical range was set at 0.05–50 μm . The precision (%CV) and accuracy (%NOM) observed were 2.3–7.2 and 88.2–96.4%. In vitro metabolism studies were performed in rat liver microsomes and results showed moderate cytochrome P450 affinity (K_m = 10.1 μm ) and a low metabolic stability of xylazine with a half‐life of 4.1 min in rat liver microsomes. Five phase 1 metabolites were observed. The main metabolite observed was an oxidation of the thiazine moiety at m/z 235 and, to a lesser extent, we observed the formation of N‐(2,6‐dimethylphenyl)thiourea at m/z 181 and three distinctive hydroxylated metabolites at m/z 237. Further experiments with ketamine and ketoconazole strongly supported that the metabolism of xylazine to its main metabolite is mediated by CYP3A in rat liver microsomes. Copyright © 2013 John Wiley & Sons, Ltd.     

Innovative determination of polar organophosphonate pesticides based on high‐resolution Orbitrap mass spectrometry

The determination of compounds showing a very low molecular weight (i.e. < 200 Da) can be complicated when low‐resolution mass spectrometry is used in the selected‐reaction monitoring mode, since the possible number of product ions is reduced and the obtained reactions are not selective enough to overcome background noise and/or matrix interferences. In this study, the use of high‐resolution mass spectrometry based on Exactive Orbitrap was applied for the determination of a group of polar organophosphonate pesticides and transformation products (TPs), which show the aforementioned features, in agricultural soils. Namely, glyphosate, glufosinate, ethephon and their TPs, aminomethyl phosphonic acid (AMPA), 3‐methylphosphinicopropionic acid, N‐acetyl‐glufosinate and 2‐hydroxyethylphosphonic acid were analyzed. The [M‐H]^‐ ions 168.00564, 180.04202, 142.96593, 110.00016, 151.01547, 222.05259 and 124.99982 were used, respectively, for the detection and identification of the compounds. Confirmation was carried out by using accurate mass measurements of ion fragments for each compound, from neutral losses of CO₂, H₂O and H₂CO (formaldehyde). Furthermore, the recently reported tool, relative isotopic mass defect (RΔm), was also used to support the confirmation protocol. The optimized method was fully validated at low levels, including the estimation of a not commonly used parameter: the limit of confirmation (LOC). This LOC is expressed as the lowest concentration of compound that can be confirmed using a fragment or the RΔm, and it ranged from 10 to 50 µg kg^?1 for all compounds. All the data was obtained in a single injection. Finally, the method was applied to real soil samples, and glyphosate and AMPA were found at 265 µg kg^?1 and 105 µg kg^?1, respectively. Copyright © 2012 John Wiley & Sons, Ltd.     

Detection and characterization of ticlopidine conjugates in rat bile using high‐resolution mass spectrometry: applications of various data acquisition and processing tools

Ticlopidine, an antiplatelet drug, undergoes extensive oxidative metabolism to form S‐oxide, N‐oxide, hydroxylated and dealkylated metabolites. However, metabolism of ticlopidine via conjugation has not been thoroughly investigated. In this study, multiple data acquisition and processing tools were applied to the detection and characterization of ticlopidine conjugates in rat bile. Accurate full‐scan mass spectrometry (MS) and collision‐induced dissociation (CID) MS/MS data sets were recorded using isotope pattern‐dependent acquisition on an LTQ/Orbitrap system. In addition, mass spectral data from online H/D exchanging and high collision energy dissociation (HCD) were recorded. Data processes were carried out using extracted ion chromatography (EIC), mass defect filter (MDF) and isotope pattern filter (IPF). The total ion chromatogram displayed a few major conjugated metabolites and many endogenous components. Profiles from EIC and IPF processes exhibited multiple conjugates with no or minimal false positives. However, ticlopidine conjugates that were not predictable or lost a chorine atom were not found by EIC or IPF, respectively. MDF was able to detect almost all of ticlopidine conjugates although it led to a few more false positives. In addition to CID spectra, data from HCD, H/D exchanging experiments and isotope pattern simulation facilitated structural characterization of unknown conjugates. Consequently, 20 significant ticlopidine conjugates, including glucuronide, glutathione, cysteinylglycine, cysteine and N‐acetylcysteine conjugates, were identified in rat bile, a majority of which are associated with bioactivation and not previously reported. This study demonstrates the utility and limitation of various high‐resolution MS‐based data acquisition and processing techniques in detection and characterization of conjugated metabolites. Copyright © 2013 John Wiley & Sons, Ltd.     

Assessment of tandem mass spectrometry and high‐resolution mass spectrometry for the analysis of bupivacaine in plasma

Triple quadrupole mass spectrometers coupled with high performance liquid chromatography are workhorses in quantitative bioanalyses. They provide substantial benefits including reproducibility, sensitivity and selectivity for trace analysis. Selected reaction monitoring allows targeted assay development but datasets generated contain very limited information. Data mining and analysis of nontargeted high‐resolution mass spectrometry profiles of biological samples offer the opportunity to perform more exhaustive assessments, including quantitative and qualitative analysis. The objectives of this study were to test method precision and accuracy, to statistically compare bupivacaine drug concentration in real study samples and to verify if high‐resolution and accurate mass data collected in scan mode can actually permit retrospective data analysis, more specifically, extract metabolite related information. The precision and accuracy data presented using both instruments provided equivalent results. Overall, the accuracy ranged from 106.2 to 113.2% and the precision observed was from 1.0 to 3.7%. Statistical comparisons using a linear regression between both methods revealed a coefficient of determination (R²) of 0.9996 and a slope of 1.02, demonstrating a very strong correlation between the two methods. Individual sample comparison showed differences from ?4.5 to 1.6%, well within the accepted analytical error. Moreover, post‐acquisition extracted ion chromatograms at m/z 233.1648 ± 5 ppm (M ? 56) and m/z 305.2224 ± 5 ppm (M + 16) revealed the presence of desbutyl‐bupivacaine and three distinct hydroxylated bupivacaine metabolites. Post‐acquisition analysis allowed us to produce semi‐quantitative evaluations of the concentration–time profiles for bupicavaine metabolites. Copyright © 2015 John Wiley & Sons, Ltd.     

Pharmacokinetic and tissue distribution studies of cassane diterpenoids,in rats through an ultra‐high‐performance liquid chromatography–Q exactive hybrid quadrupole–Orbitrap high‐resolution accurate mass spectrometry

Cassane diterpenoids (CA) are considered as the main active constituents of medicinal plants belonging to the Caesalpinia genus. Three cassane derivatives, bonducellpin G (BG), 7‐O‐acetyl‐bonducellpin C (7‐O‐AC) and caesalmin E (CE), isolated from Caesalpinia minax Hance seeds, showed strong anti‐inflammatory activity. In this paper, pharmacokinetics (BG, 7‐O‐AC, CE) and tissue distribution (7‐O‐AC, CE) properties were studied for the first time using a reliable, sensitive and rapid UHPLC–Q‐Orbitrap HR‐MS to develop new anti‐inflammatory agents. A novel quantitative method with full scan in positive ion mode was used to determine the contents of compounds. They were separated using acetonitrile–water (0.1% formic acid) as gradient mobile phase. The calibration curve displayed good linearity and the lower limit of quantitation was 0.005–0.02 μg/mL for all analytes. Meanwhile, the absorption, distribution, metabolism, excretion (ADME) property was predicted using PreADMET web. The pharmacokinetic parameters indicated that they were absorbed quickly, eliminated rapidly together with high blood concentration. The results of tissue distribution demonstrated that CE was distributed rapidly and widely among tissues, and stomach was the main tissue site of CE and 7‐O‐AC, followed by small intestine/liver. This study indicates that the structures and dosages of active CA should be modified to help improve the absorption rate and residence time, and the findings are helpful for the pharmaceutical design of CA derivatives.     

The determination of polycyclic aromatic hydrocarbons in human urine by high‐resolution gas chromatography–mass spectrometry

Polycyclic aromatic hydrocarbons (PAHs), organic compounds formed by at least two condensed aromatic rings, are ubiquitous environmental pollutants that are produced by incomplete combustion of organic materials. PAHs have been classified as carcinogenIC to humans by the International Agency for Research on Cancer, because they can bind to DNA, causing mutations. Therefore, the levels of PAHs in human urine can be used as an indicator for potential carcinogenesis and cell mutation. An analytical method was developed for the accurate measurement of PAHs in urine using high‐resolution gas chromatography–mass spectrometry. Urine samples were extracted by an Oasis HLB extraction cartridge after enzymatic hydrolysis with a β‐glucuronidase/arylsulfatase cocktail. The 18 PAHs were separated using an Agilent DB‐5 MS capillary column (30 m × 0.25 mm, 0.25 μm) and monitored by time‐of‐flight mass spectrometry. Under the optimized method, the linearity of calibration curves was >0.994. The limits of detection at a signal‐to‐noise ratio of 3 were 10–100 ng/L. The coefficients of variation were in the range of 0.4–9.0%. The present method was highly accurate for simultaneous determination of 18 PAHs in human urine and could be applied to monitoring and biomedical investigations to check exposure of PAHs.     

Metabolite identification of the antimalarial naphthoquine using liquid chromatography–tandem high‐resolution mass spectrometry in combination with multiple data‐mining tools

Naphthoquine (NQ) is one of important partner drugs of artemisinin‐based combination therapy (ACT), which is recommended for the treatment of uncomplicated Plasmodium falciparum. NQ shows a high cure rate after a single oral administration. It is absorbed quickly (time to peak concentration 2–4 h) and has a long elimination half‐life (255 h). However, the metabolism of NQ has not been clarified. In this work, the metabolite profiling of NQ was studied in six liver microsomal incubates (human, cynomolgus monkey, beagle dog, mini pig, rat and CD1 mouse), seven recombinant CYP enzymes (1A2, 2B6, 2C8, 2C9, 2C19, 2D6 and 3A4) and rat (plasma, urine, bile and feces) using liquid chromatography tandem high‐resolution LTQ‐Orbitrap mass spectrometry (HRMSⁿ) in conjunction with online hydrogen/deuterium exchange. The biological samples were pretreated by protein precipitation and solid‐phase extraction. For data processing, multiple data‐mining tools were applied in tandem, i.e. background subtraction and followed by mass defect filter. NQ metabolites were characterized by accurate MS/MS fragmentation characteristics, the hydrogen/deuterium exchange data and cLogP simulation. As a result, five phase I metabolites (M1–M5) of NQ were characterized for the first time. Two metabolic pathways were involved: hydroxylation and N‐oxidation. This study demonstrates that LC‐HRMSⁿ in combination with multiple data‐mining tools in tandem can be a valuable analytical strategy for rapid metabolite profiling of drugs.     

Comprehensive characterization and quantification of multiple components in Dan‐Huang‐Qu‐Yu capsule using a multivariate data processing approach based on microwave‐assisted extraction with UHPLC and Q Exactive quadrupole‐orbitrap high‐resolution mass spectrometry

Dan‐Huang‐Qu‐Yu capsule, a Chinese herbal medicine compound preparation, is widely used for chronic pelvic inflammatory disease. In this study, a rapid, selective, and sensitive microwave‐assisted extraction ultra‐high‐performance liquid chromatography‐Q Exactive quadrupole‐orbitrap high‐resolution mass spectrometry method was developed for analyzing its chemical compositions. A total of 85 compounds, including 22 flavonoids, 8 terpenoids, 5 quinones, 5 phthaleolactone, 23 organic acids, and 22 other compounds were identified from Dan‐Huang‐Qu‐Yu capsule. Among them, 35 major compounds were unambiguously detected by comparing them with reference standards and selected as quality control markers, which were simultaneously determined in Dan‐Huang‐Qu‐Yu capsule. The established method was successfully validated and applied for simultaneous determination of 35 bioactive compounds in Dan‐Huang‐Qu‐Yu capsule from ten sample batches. The quantitative data of the analytes were analyzed by principal component analysis for quality assessment of Dan‐Huang‐Qu‐Yu capsule. Six compounds (e.g., astragaloside IV, salvianolic acid B, ellagic acid, chlorogenic acid, N‐butylidenephthalide, and luteolin) were screened out and regarded as chemical markers for quality control of Dan‐Huang‐Qu‐Yu capsule. The established method has been proved to be a novel and useful tool for rapid research of Dan‐Huang‐Qu‐Yu capsule. This research will provide reference for the scientific research of traditional Chinese medicines.     

Metabolite identification of the antimalarial piperaquine in vivo using liquid chromatography–high‐resolution mass spectrometry in combination with multiple data‐mining tools in tandem

Artemisinin‐based combination therapy is widely used for the treatment of uncomplicated Plasmodium falciparum malaria, and piperaquine (PQ) is one of important partner drugs. The pharmacokinetics of PQ is characterized by a low clearance and a large volume of distribution; however, metabolism of PQ has not been thoroughly investigated. In this work, the metabolite profiling of PQ in human and rat was studied using liquid chromatography tandem high‐resolution LTQ‐Orbitrap mass spectrometry (HRMS). The biological samples were pretreated by solid‐phase extraction. Data processes were carried out using multiple data‐mining techniques in tandem, i.e., isotope pattern filter followed by mass defect filter. A total of six metabolites (M1–M6) were identified for PQ in human (plasma and urine) and rat (plasma, urine and bile). Three reported metabolites were also found in this study, which included N‐oxidation (M1, M2) and carboxylic products (M3). The subsequent N‐oxidation of M3 resulted in a new metabolite M4 detected in urine and bile samples. A new metabolic pathway N‐dealkylation was found for PQ in human and rat, leading to two new metabolites (M5 and M6). This study demonstrated that LC‐HRMSⁿ in combination with multiple data‐mining techniques in tandem can be a valuable analytical strategy for rapid metabolite profiling of drugs. Copyright © 2016 John Wiley & Sons, Ltd.     

Pharmacokinetic studies of soyalkaloid A from Portulaca oleracea L. using ultra high‐performance liquid chromatography electrospray ionization quadrupole–time of flight mass spectrometry and its antioxidant activity

Soyalkaloid A was isolated from Portulaca oleracea L. for the first time in our laboratory and then a rapid and sensitive ultra‐high‐performance liquid chromatography electrospray ionization quadrupole–time of flight mass spectrometry (UHPLC–ESI–Q–TOF/MS) method with hesperidin as internal standard (IS) was developed and validated to investigate the pharmacokinetics of soyalkaloid A in rats after oral and intravenous administrations. The analysis was achieved on an Agilent Zorbax Eclipse Plus C₁₈ Column (2.1 × 50 mm, 1.8 μm) by elution with acetonitrile and water (containing 0.1% formic acid), at a flow rate of 0.3 mL/min. The MS analysis was performed in the positive ion mode with monitored ion m/z 227.0814 [M + H]⁺ and 611.1971 [M + H]⁺ for soyalkaloid A and IS, respectively. The linear range was established over the concentration range 7.5–6000 ng/mL (r = 0.9951). The intra‐ and inter‐assay accuracy and precision were between ?4.86‐4.49 and 1.93–9.66, respectively. The lower limits of detection and quantitation observed were 2.1 and 7.4 ng/mL, respectively. The rapid, sensitive and specific UHPLC–ESI–Q–TOF/MS method was successfully applied to a pharmacokinetic study of soyalkaloid A. Moreover, its antioxidant was studied via a 1,1‐diphenyl‐2‐picryl‐hydrazyl radical scavenging assay, the IC₅₀ value being 20.73 ± 0.51 μM.     

Stereoselective determination of dufulin in watermelon under field conditions using chiral ultra high performance liquid chromatography with high‐resolution mass spectrometry

An effective and sensitive chiral analytical method was established to investigate the stereoselective dissipation of rac‐dufulin in watermelon using ultra high performance liquid chromatography with a superchiral S‐OD chiral column (4.6 × 150 mm i.d., 5 μm) coupled with high‐resolution mass spectrometry. To optimize the pretreatment method for detecting rac‐dufulin in the three matrixes, different extraction solvents, extractant volumes, extraction times, and absorbents were investigated to improve extraction efficiency. Moreover, analysis of variance was used to perform method validation for determination of the two dufulin enantiomers in the three matrixes. Using the optimized method, good linearity was obtained (determination coefficient > 0.999). The limits of detection and quantification of the two dufulin enantiomers in soil, watermelon, and pulp were 0.15 and 0.5 μg/kg, respectively. The average recoveries of the two enantiomers in the three matrixes at four spiked levels ranged from 75.0 to 107.8%, with intra‐ and inter‐day relative standard deviations of 0.4–10.4%. In field trials, the R enantiomer was preferentially dissipated in watermelon. These method validation results confirmed that the developed method was convenient and reliable for the stereoselective determination of enantiomers of rac‐dufulin in watermelon.     

Tentative identification of polar and mid‐polar compounds in extracts from wine lees by liquid chromatography–tandem mass spectrometry in high‐resolution mode

Sustainable agriculture has a pending goal in the revalorization of agrofood residues. Wine lees are an abundant residue in the oenological industry. This residue, so far, has been used to obtain tartaric acid or pigments but not for being qualitatively characterized as a source of polar and mid‐polar compounds such as flavonoids, phenols and essential amino acids. Lees extracts from 11 Spanish wineries have been analyzed by liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS) in high resolution mode. The high‐resolution power of LC–MS/MS has led to the tentative identification of the most representative compounds present in wine lees, comprising primary amino acids, anthocyans, flavanols, flavonols, flavones and non‐flavonoid phenolic compounds, among others. Attending to the profile and content of polar and mid‐polar compounds in wine lees, this study underlines the potential of wine lees as an exploitable source to isolate interesting compounds. Copyright © 2015 John Wiley & Sons, Ltd.     

Targeted and nontargeted screening and identification of 50 antihypertensive adulterants in dietary supplements and herbal medicines using quadrupole–orbitrap high resolution mass spectrometry with compound database

In the present work, a novel database of drug compounds and a rapid screening method based on ultra‐high performance liquid chromatography coupled to high resolution orbitrap mass spectrometry were developed and applied in the screening and identification of targeted and nontargeted antihypertensive adulterants in dietary supplements and herbal medicines. The established screening database includes retention time, exact mass, fragments, isotopic pattern, and MS² spectra library of the target compounds and thus provides automated search and identification of the targets with a single injection. The nontargeted compounds in the samples are identified through the full MS scan and MS² data by using the Chemspider database and the data analysis in XCalibur, MassFrontier and TraceFinder software. In addition, this method possesses excellent quantitative capacity. The novel approach was applied to 65 batches of samples that are claimed as “all‐natural” products having the antihypertensive function, among which nine batches were found to be positive. Multiple targeted and nontargeted antihypertensive adulterants were detected at levels ranging from 2.8 to 27.9 mg/g. The novel database and screening method demonstrated herein will be promising and powerful tools for rapid screening of antihypertensive adulterants in dietary supplements and herbal medicines.     

Novel strategy for the determination of illegal adulterants in health foods and herbal medicines using high‐performance liquid chromatography with high‐resolution mass spectrometry

The detection, confirmation, and quantification of multiple illegal adulterants in health foods and herbal medicines by using a single analytical method are a challenge. This paper reports on a new strategy to meet this challenge by employing high‐performance liquid chromatography coupled with high‐resolution mass spectrometry and a mass spectral tree similarity filter technique. This analytical method can rapidly collect high‐resolution, high‐accuracy, optionally multistage mass data for compounds in samples. After a preliminary screening by retention time and high‐resolution mass spectral data, known illegal adulterants can be detected. The mass spectral tree similarity filter technique has been applied to rapidly confirm these adulterants and simultaneously discover unknown ones. By using full‐scan mass spectra as stem and data‐dependent subsequent stage mass spectra to form branches, mass spectrometry data from detected compounds are converted into mass spectral trees. The known or unknown illegal adulterants in the samples are confirmed or discovered based on the similarity between their mass spectral trees and those of the references in a library, and they are finally quantified against standard curves. This new strategy has been tested by using 50 samples, and the illegal adulterants were rapidly and effectively detected, confirmed and quantified.     

Screening for pharmaco‐toxicologically relevant compounds in biosamples using high‐resolution mass spectrometry: a ‘metabolomic’ approach to the discrimination between isomers

High‐resolution mass spectrometry (HRMS) enables the identification of a chemical formula of small molecules through the accurate measurement of mass and isotopic pattern. However, the identification of an unknown compound starting from the chemical formula requires additional tools: (1) a database associating chemical formulas to compound names and (2) a way to discriminate between isomers. The aim of this present study is to evaluate the ability of a novel ‘metabolomic’ approach to reduce the list of candidates with identical chemical formula. Urine/blood/hair samples collected from real positive cases were submitted to a screening procedure using ESI‐MS‐TOF (positive‐ion mode) combined with either capillary electrophoresis or reversed phase liquid chromatography (LC). Detected peaks were searched against a Pharmaco/Toxicologically Relevant Compounds database (ca 50 500 compounds and phase I and phase II metabolites) consisting of a subset of PubChem compounds and a list of candidates was retrieved. Then, starting from the mass of unknown, mass shifts corresponding to pre‐defined biotransformations (e.g. demethylation, glucuronidation, etc.) were calculated and corresponding mass chromatograms were extracted from the total ion current (TIC) in order to search for metabolite peaks. For each candidate, the number of different functional groups in the molecule was automatically calculated using E‐Dragon software (Talete srl, Milan, Italy). Then, the presence of metabolites in the TIC was matched with functional groups data in order to exclude candidates with structures not compatible with observed biotransformations (e.g. loss of methyl from a structure not bearing methyls). The procedure was tested on 108 pharmaco‐toxicologically relevant compounds (PTRC) and their phase I metabolites were detected in real positive samples. The mean list length (MLL) of candidates retrieved from the database was 7.01 ± 4.77 (median, 7; range, 1–28) before the application of the ‘metabolomic’ approach, and after the application it was reduced to 4.08 ± 3.11 (median 3, range 1–17). HRMS allows a much broader screening for PTRC than other screening approaches (e.g. library search on mass spectra databases). The ‘metabolomic’ approach enables the reduction of the list of candidate isomers. Copyright © 2009 John Wiley & Sons, Ltd.