High-temperature ultra-performance liquid chromatography coupled to hybrid quadrupole time-of-flight mass spectrometry applied to ibuprofen metabolites in human urine

The application of sub-2 microm porous particle liquid chromatography (LC) operated at elevated temperatures, coupled with time-of-flight mass spectrometry (MS), to the separation and identification of metabolites of ibuprofen present in human urine following oral administrations is illustrated. The LC/MS system generated a high-resolution analytical separation that, with an analysis time of 20 min, provided a peak capacity in the order of ca. 350. Using this system a total of nine glucuronides of the drug and its metabolites were detected, including a number of isomeric acyl glucuronides of ibuprofen itself, a side-chain-oxidized carboxylic acid acyl glucuronide and a number of acyl glucuronides of various hydroxylated metabolites. The identities of the metabolites were confirmed by their accurate mass values and the presence of the common fragment ions from ibuprofen.     

Ultra-performance liquid chromatography coupled to quadrupole-orthogonal time-of-flight mass spectrometry

Ultra-performance liquid chromatography (UPLC) utilizes sub-2 microm particles with high linear solvent velocities to effect dramatic increases in resolution, sensitivity and speed of analysis. The reduction in particle size to below 2 microm requires instrumentation that can operate at pressures in the 6000-15,000 psi range. The typical peak widths generated by the UPLC system are in the order of 1-2 s for a 10-min separation. In the present work this technology has been applied to the study of in vivo drug metabolism, in particular the analysis of drug metabolites in bile. The reduction in peak width significantly increases analytical sensitivity by three- to five-fold, and the reduction in peak width, and concomitant increase in peak capacity, significantly reduces spectral overlap resulting in superior spectral quality in both MS and MS/MS modes. The application of UPLC/MS resulted in the detection of additional drug metabolites, superior separation and improved spectral quality.     

Identification and structural characterization of biodegradation products of atenolol and glibenclamide by liquid chromatography coupled to hybrid quadrupole time-of-flight and quadrupole ion trap mass spectrometry

In this paper we report about the biodegradation of the beta-blocker atenolol and the hypoglycaemic agent glibenclamide. The biodegradation tests were performed in batch reactors under aerobic conditions, using as inocculums sewage sludge from a conventional activated sludge treatment and a laboratory-scale membrane bioreactor. Pharmaceuticals were used as sole carbon sources, spiked at 50ng/L and 10mg/L concentrations. Quadrupole time-of-flight mass spectrometry coupled to ultra-high-pressure liquid chromatograph was used for the screening and the structural elucidation of biodegradation products. A microbial metabolite of atenolol with [M+H](+) at 268 was detected in the positive electrospray ionization mode. This new compound was determined to be a product of microbial hydrolysis of the amide of the parent compound. Biodegradation of glibenclamide by activated sludge proceeded via bacterial hydroxylation of the cyclohexyl ring, which resulted in formation of metabolite with a protonated molecule, [M+H](+)=510. MS(3) experiments performed by hybrid quadrupole linear ion trap (QqLIT) mass spectrometry coupled to high-performance liquid chromatography enabled further structural elucidation of the identified metabolites. Moreover, the highly sensitive QqLIT instrument in the MRM mode enabled the detection of parent compounds and one of the microbial metabolites identified in real wastewater samples. The methodology used in this study permitted for the first time the identification and detection of biodegradation product of beta-blocker atenolol in real wastewater samples.     

A comprehensive study of celastrol metabolism in vivo and in vitro using ultra-high-performance liquid chromatography coupled with hybrid triple quadrupole time-of-flight mass spectrometry

Celastrol has attracted great attention owing to its anti-arthritis, antioxidant, and anticancer activities. Nevertheless, its metabolism in vivo (rats) and in vitro (rat liver microsomes and intestinal flora) has not been comprehensively characterized. In this study, ultra-high-performance liquid chromatography coupled with hybrid triple quadrupole time-of-flight mass spectrometry was used as a rapid and sensitive approach for studying the metabolism of celastrol in vivo and in vitro. A total of 43 metabolites were identified and characterized. These include 26 metabolites in vivo, and 28 metabolites in vitro (nine metabolites in rat liver microsomes and 24 metabolites in rat intestinal flora). Additionally, the celastrol-biotransformation capacity of the intestinal tract was confirmed to exceed that of the liver. Furthermore, the metabolic profile of celastrol is summarised. The information obtained from this study may provide a basis for understanding the pharmacological mechanisms of celastrol and will be beneficial for clinical applications.     

Screening of antibiotics in surface and wastewater samples by ultra-high-pressure liquid chromatography coupled to hybrid quadrupole time-of-flight mass spectrometry

The potential of ultra-high-pressure liquid chromatography (UHPLC) coupled to hybrid quadrupole time-of-flight mass spectrometry (QTOF–MS) for the screening and confirmation of antibiotics in water samples is illustrated in this paper. UHPLC presents several advantages over conventional liquid chromatography as it generates narrow peaks (increasing peak height and improving sensitivity) and reduces chromatographic runs. Regarding QTOF–MS, its increased mass resolution, high sensitivity in full-spectrum acquisition mode and mass accuracy, in both MS and MS/MS modes, make this technique ideal for the detection and reliable confirmation of organic contaminants in environmental samples. After a solid-phase extraction using Oasis HLB cartridges, UHPLC–QTOF–MS has been applied in this work to several types of water samples (surface water and influent and effluent wastewaters). Several antibiotics were found in the samples, such as ofloxacin, ciprofloxacin, clarythromycin or erythromycin, among others. Moreover, the full spectrum data provided by TOF–MS acquisition has enabled searching for many other pharmaceuticals that could be present in the samples in a “post-target” way. This approach has allowed the detection and confirmation of paracetamol, omeprazole and codeine, among others. UPLC–QTOF–MS has been shown as an attractive and efficient hyphenated technique for the rapid detection and confirmation of pharmaceuticals in water with very little sample handling.     

Rapid screening and characterization of metabolites from a marine-derived actinomycete by high-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight mass spectrometry

A rapid and reliable method has been optimized and established for the analysis of the metabolites from a marine actinomycete by high‐performance liquid chromatography coupled with electrospray ionization quadrupole time‐of‐flight mass spectrometry (HPLC/QTOF MS/MS). From MS/MS spectra, the product ions of [M + H]⁺ were recorded to provide abundant structural information of the mother nucleus and peptide moieties. Using the QTOF MS/MS and in‐source collision‐induced dissociation (in‐source CID) techniques, three main metabolites including actinomycin D, actinomycin V and actinomycin I were determined and characterized by elemental compositions of precursor and product ions (<7 ppm). Additionally, this method provided information about the compositions of the peptide residues and the sequences of the amino acid from a series of fragment ions. It proved useful for the identi?cation of the metabolites in marine samples which have similar structures especially when there were no reference compounds available. Copyright © 2010 John Wiley & Sons, Ltd.     

High-throughput intracellular pteridinic profiling by liquid chromatography–quadrupole time-of-flight mass spectrometry

Pteridines are a diverse family of endogenous metabolites that may serve as useful diagnostic biomarkers for disease. While many preparative and analytical techniques have been described for analysis of selected pteridines in biological fluids, broad intracellular pteridine detection remains a significant analytical challenge. In this study, a novel, specific and sensitive extraction and high performance liquid chromatography–quadrupole time-of-flight mass spectrometry (HPLC–QTOF MS) method was developed to simultaneously quantify seven intracellular pteridines and monitor 18 additional, naturally-occurring intracellular pteridines. The newly developed method was validated through evaluation of spiked recoveries (84.5–109.4%), reproducibility (2.1–5.4% RSD), method detection limits (0.1–3.0 μg L⁻¹) and limits of quantitation (0.1–1 μg L⁻¹), and finally application to non-small cell lung cancer A549 cells. Twenty-three pteridine derivatives were successfully detected from cell lysates with an average RSD of 12% among culture replicates. Quantified intracellular pteridine levels ranged from 1 to 1000 nM in good agreement with previous studies. Finally, this technique may be applied to cellular studies to generate new biological hypotheses concerning pteridine physiological and pathological functions as well as to discovery new pteridine-based biomarkers.     

Comprehensive blood plasma lipidomics by liquid chromatography/quadrupole time-of-flight mass spectrometry

A lipidomics strategy, combining high resolution reversed-phase liquid chromatography (RPLC) with high resolution quadrupole time-of-flight mass spectrometry (QqTOF), is described. The method has carefully been assessed in both a qualitative and a quantitative fashion utilizing human blood plasma. The inherent low technical variability associated with the lipidomics method allows to measure 65% of the features with an intensity RSD value below 10%. Blood plasma lipid spike-in experiments demonstrate that relative concentration differences smaller than 25% can readily be revealed by means of a t-test. Utilizing an advanced identification strategy, it is shown that the detected features mainly originate from (lyso-)phospholipids, sphingolipids, mono-, di- and triacylglycerols and cholesterol esters. The high resolution offered by the up-front RPLC step further allows to discriminate various isomeric species associated with the different lipid classes. The added value of utilizing a Jetstream electrospray ionization (ESI) source over a regular ESI source in lipidomics is for the first time demonstrated. In addition, the application of ultra high performance LC (UHPLC) up to 1200 bar to extend the peak capacity or increase productivity is discussed.     

Identification of metabolites of Ginkgolide B in vivo and in vitro using ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry

Ginkgolide B is a dietary diterpene with multiple pharmacological activities. However, current research on ginkgolide B is not comprehensive. The current study analyzed the metabolic profile of ginkgolide B in vivo and in vitro using ultra-high-performance liquid chromatography quadrupole time-of-flight mass spectrometry. To detect and identify the different metabolites in ginkgolide B, a novel data processing method was used as an assistant tool. A total of 53 different metabolites of ginkgolide B (38 phase I metabolites and 15 phase II metabolites) were detected relative to blank samples. The biotransformation route of ginkgolide B was identified as oxidation, dehydroxylation, hydrogenation, decarbonylation, demethylation, sulfate conjugation, glucose conjugation, methylation, and acetylation. The current study demonstrated a method for rapidly detecting and identifying metabolites and provided useful information to further characterize the pharmacology and mechanism of ginkgolide B. A method for the analysis of other diterpene metabolic components in vivo and in vitro was also established.     

Characterization of chemical components of Periplocae Cortex and their metabolites in rats using ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry

Periplocae Cortex, named Xiang-Jia-Pi in China, has been widely used to treat autoimmune diseases, especially rheumatoid arthritis. However, the in vivo substances of Periplocae Cortex remain unknown yet. In this study, an ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry was used for profiling the chemical components and related metabolites of Periplocae Cortex. A total of 98 constituents were identified or tentatively characterized in Periplocae Cortex: 42 C21 steroidal glycosides, 10 cardiac glycosides, 23 organic acids, 4 aldehydes, 7 triterpenes, and 12 other types. Among them, 18 components were unambiguously identified by comparison with reference standards. In addition, 176 related xenobiotics (34 prototypes and 142 metabolites) were screened out and characterized in rats’ biosamples (plasma, urine, bile, and feces) after the oral administration of Periplocae Cortex. Moreover, the metabolic fate of periplocoside S-4a, a C21 steroidal glycoside, was proposed for the first time. In summary, phase II reactions (methylation, glucuronidation, and sulfation), phase I reactions (hydrolysis reactions, oxygenation, and reduction), and their combinations were the predominant metabolic reactions of Periplocae Cortex in rat. It is the first report to reveal the in vivo substances and metabolism feature of Periplocae Cortex. This study also provided meaningful information for further pharmacodynamics study of Periplocae Cortex, as well as its quality control research.     

Identification and characterization of impurities of tetracosactide by capillary electrophoresis and liquid chromatography coupled to time-of-flight mass spectrometry

Tetracosactide is a synthetic peptide analogue of the human adrenocorticotropic hormone that stimulates the production of cortisol in the adrenal cortex. The medical use of the compound is primarily the diagnosis of the adrenal cortex function. In order to characterize impurities of the drug, tetracosactide samples were analysed by both liquid chromatography and capillary electrophoresis coupled to a quadrupole time-of-flight mass spectrometer. The identification of the impurities was carried out based on accurate mass determination and fragment ion spectra. The presence of several peptides of lower and higher masses than tetracosactide could be shown, including N- and C-terminally truncated peptides as well as peptides which still contained protecting groups or additional amino acids. Furthermore, a semi-quantitative estimation of the relative amounts of the impurities in different samples as well as a commercial preparation revealed that the number and the type of the impurities varied between the samples. Comparing the selectivity of liquid chromatography and capillary electrophoresis regarding the separation of tetracosactide impurities, it can be stated that capillary electrophoresis showed a higher suitability for the separation of tetracosactide fragments (smaller peptides) while the larger peptides, i.e. those wearing protecting groups, were separated more efficiently by liquid chromatography.     

Chemical profiling of Qingfei Paidu Decoction by triplex off-line two-dimensional liquid chromatography coupled with quadrupole time-of-flight mass spectrometry

Qingfei Paidu Decoction is a Chinese medicine formula that has been proved effective in the treatment of coronavirus disease 2019. However, the comprehensive separation and characterization of Qingfei Paidu Decoction are of a great challenge due to the diversity of chemical components in a wide range of polarity. In this study, a triplex off-line two-dimensional liquid chromatography coupled with quadrupole time-of-flight mass spectrometry is developed for the analysis of Qingfei Paidu Decoction. One reversed-phase liquid chromatography×hydrophilic interaction liquid chromatography system and two reversed-phase liquid chromatography×reversed phase liquid chromatography systems were constructed to separate polar components and weak-polar components in Qingfei Paidu Decoction, respectively. Benefiting from the good orthogonality of two-dimensional liquid chromatography and high sensitivity of quadrupole time-of-flight MS, chemical components with different polarities and content were discovered. A total of 749 peaks were detected in positive and negative ionization mode and presented as a four-dimensional data plot. Meanwhile, 498 compounds belonging to 14 categories were tentatively identified. These results provide good supplementary to elucidate the material basis of Qingfei Paidu Decoction. The triplex off-line two-dimensional liquid chromatography-quadrupole time-of-flight mass spectrometry strategy can be a powerful and efficient tool for the separation and characterization of chemical substances in traditional Chinese medicine formulas.     

Identification of metabolites of geniposide in rat urine using ultra-performance liquid chromatography combined with electrospray ionization quadrupole time-of-flight tandem mass spectrometry

Geniposide, an iridoid glycoside, is an important and characteristic compound in the fruits of Gardenia jasminoides Ellis, a commonly used medicinal herb in Chinese traditional and folk medicine for the treatment of inflammation and jaundice. However, few studies have been carried out on the metabolism of geniposide. In this study, we have established a rapid and sensitive method using ultra‐performance liquid chromatography coupled with electrospray ionization quadrupole time‐of‐flight tandem mass spectrometry (UPLC/ESI‐QTOF‐MS) for analysis of the metabolic profile of geniposide in rat urine after oral administration. A total of ten metabolites were detected and identified by comparing their fragmentation patterns with that of geniposide using Metabolynx? and MassFragment? software tools. The results revealed that the principal metabolism pathways of geniposide in rat occurred after deglycosylation of the irdoid glycoside take place and this is followed by glucuronidation and the pyran‐ring cleavages. The major metabolite, the glucuronic acid conjugate of genipin as observed in vivo, was further confirmed by the in vitro enzymatic study. The results of this work have demonstrated the feasibility of the UPLC/ESI‐QTOF‐MS approach for rapid and reliable characterization of metabolites from iridoid compounds. Copyright © 2011 John Wiley & Sons, Ltd.     

Elucidating the structure of carbon nanoparticles by ultra-performance liquid chromatography coupled with electrospray ionisation quadrupole time-of-flight tandem mass spectrometry

A fast and accurate ultra-performance liquid chromatography coupled with electrospray ionisation quadrupole time-of-flight tandem mass spectrometry (UPLC-ESI-Q-TOF-MS/MS) method was developed for the separation and structural elucidation of fluorescent carbon nanoparticles (CNP). The CNP was synthesised from microwave-assisted pyrolysis of citric acid (CA) and 1,2-ethylenediamine (EDA). By using UPLC separation, the CNP product was well separated into ten fractions within 4.0 min. Based on high-accuracy MS and MS/MS analyses, the CNP species were revealed to display six kinds of chemical formulas, including (C₁₀H₂₀N₄O₅)_n, (C₈H₁₂N₂O₅)_n, (C₁₆H₂₂N₄O₉)_n, (C₆H₈O₇)_n, (C₁₄H₁₈N₂O₁₁)_n, and (C₁₄H₁₆N₂O₁₀)_n. In particular, our study revealed for the first time that the CNP species exist as supramolecular clusters with their individual monomers units linked together through non-covalent bonding forces. These findings clearly indicated the usefulness of UPLC-ESI-Q-TOF-MS/MS in identifying the chemical composition of CNP product. It is anticipated that our proposed methodology can be applied to study the structure-property relationships of CNP, facilitating in the production of CNP with desirable spectral features.     

Quantification of glucuronidated and sulfated steroids in human urine by ultra-high pressure liquid chromatography quadrupole time-of-flight mass spectrometry

The urinary steroid profile is constituted by anabolic androgenic steroids, including testosterone and its relatives, that are extensively metabolized into phase II sulfated or glucuronidated steroids. The use of liquid chromatography coupled to mass spectrometry (LC-MS) is an issue for the direct analysis of conjugated steroids, which can be used as urinary markers of exogenous steroid administration in doping analysis, without hydrolysis of the conjugated moiety. In this study, a sensitive and selective ultra high-pressure liquid chromatography coupled to quadrupole time-of-flight mass spectrometer (UHPLC-QTOF-MS) method was developed to quantify major urinary metabolites simultaneously after testosterone intake. The sample preparation of the urine (1 mL) was performed by solid-phase extraction on Oasis HLB sorbent using a 96-well plate format. The conjugated steroids were analyzed by UHPLC-QTOF-MS^E with a single-gradient elution of 36 min (including re-equilibration time) in the negative electrospray ionization mode. MS^E analysis involved parallel alternating acquisitions of both low- and high-collision energy functions. The method was validated and applied to samples collected from a clinical study performed with a group of healthy human volunteers who had taken testosterone, which were compared with samples from a placebo group. Quantitative results were also compared to GC-MS and LC-MS/MS measurements, and the correlations between data were found appropriate. The acquisition of full mass spectra over the entire mass range with QTOF mass analyzers gives promise of the opportunity to extend the steroid profile to a higher number of conjugated steroids.     

Quantification of clenbuterol in equine plasma,urine and tissue by liquid chromatography coupled on-line with quadrupole time-of-flight mass spectrometry

Clenbuterol (CBL) is a potent beta(2)-adrenoceptor agonist used for the management of respiratory disorders in the horse. The detection and quantification of CBL can pose a problem due to its potency, the relatively low dose administered to the horse, its slow clearance and low plasma concentrations. Thus, a sensitive method for the quantification and confirmation of CBL in racehorses is required to study its distribution and elimination. A sensitive and fast method was developed for quantification and confirmation of the presence of CBL in equine plasma, urine and tissue samples. The method involved liquid-liquid extraction (LLE), separation by liquid chromatography (LC) on a short cyano column, and pseudo multiple reaction monitoring (pseudo-MRM) by electrospray ionization quadrupole time-of-flight tandem mass spectrometry (ESI-QTOF-MS/MS). At very low concentrations (picograms of CBL/mL), LLE produced better extraction efficiency and calibration curves than solid-phase extraction (SPE). The operating parameters for electrospray QTOF and yield of the product ion in MRM were optimized to enhance sensitivity for the detection and quantification of CBL. The quantification range of the method was 0.013-10 ng of CBL/mL plasma, 0.05-20 ng/0.1 mL of urine, and 0.025-10 ng/g tissue. The detection limit of the method was 13 pg/mL of plasma, 50 pg/0.1 mL of urine, and 25 pg/g of tissue. The method was successfully applied to the analysis of CBL in plasma, urine and various tissue samples, and in pharmacokinetic (PK) studies of CBL in the horse. CBL was quantified for 96 h in plasma and 288 h in urine post-administration of CLB (1.6 micro g/kg, 2 x daily x 7 days). This method is useful for the detection and quantification of very low concentrations of CBL in urine, plasma and tissue samples.     

Characterization of carbofuran photodegradation by-products by liquid chromatography/hybrid quadrupole time-of-flight mass spectrometry

The structural elucidation of by-products arising from carbofuran photodegradation using a high-pressure UV lamp has been investigated by liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) employing a quadrupole time-of-flight mass spectrometer. Exact mass measurements of the [M + H]+ ions of the by-products and of product ions allowed the elemental formulae and related structures of seven photodegradation by-products (resulting, respectively, from photo-Fries rearrangement, hydroxylation of the benzene ring, oxidation of the 2,3-dihydrobenzofuran ring, cleavage of the carbamate group, hydrolysis of the ether group and the newly observed radical coupling and decarboxylation processes) to be determined confidently. Accurate mass measurements of product ions allowed ambiguities to be removed concerning neutral losses having the same nominal mass, namely CO and C2H4, allowing the fragmentation patterns to be rationalized.