Liquid chromatography–electrochemistry–mass spectrometry of polycyclic aromatic hydrocarbons

An efficient method for fast elucidation of the electrochemical reactions of polycyclic aromatic hydrocarbons (PAH) has been set up by applying post-column electrochemistry in liquid chromatography–mass spectrometry (LC–MS). With this set-up strong improvement of sensitivity in the LC–MS analysis of PAH is observed. Due to their low redox potentials, the non-polar PAH are converted into the respective radical cations, which may further react with constituents of the mobile phase and in additional electrochemical oxidation steps. Among other products, mono-, di-, and trioxygenated species are observed in aqueous solutions, alkoxylated compounds in alcohols, and solvent adducts in the presence of acetonitrile. While more different products are observed by using atmospheric pressure chemical ionization in the positive-ion mode (APCI(+)), the deprotonation of hydroxylated species results in very clear spectra in the negative-ion mode (APCI(–)). Deuterated PAH and deuterated solvents were used to gain additional information on the formation of the reaction products.     

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.     

Impurity profiling of acetylspiramycin by liquid chromatography–ion trap mass spectrometry

Investigation of acetylspiramycin (ASPM) and its related substances was carried out using a reversed-phase liquid chromatography/tandem mass spectrometry method. The identification of impurities in the ASPM complex was performed with a quadrupole ion trap mass spectrometer, with an electrospray ionization (ESI) source in the positive ion mode which provides MSⁿ capability. A total of 83 compounds were characterized in commercial samples, among which 31 impurities that had never been reported and 31 partially characterized impurities were deduced using the collision-induced dissociation (CID) spectra of major ASPM components as templates. Most of the major impurities arise from the starting materials and the synthesis process. This work provides very useful information for quality control of ASPM and evaluation of its synthesis process.     

Tutorial review on validation of liquid chromatography–mass spectrometry methods: Part I

This is the part I of a tutorial review intending to give an overview of the state of the art of method validation in liquid chromatography mass spectrometry (LC–MS) and discuss specific issues that arise with MS (and MS/MS) detection in LC (as opposed to the “conventional” detectors). The Part I briefly introduces the principles of operation of LC–MS (emphasizing the aspects important from the validation point of view, in particular the ionization process and ionization suppression/enhancement); reviews the main validation guideline documents and discusses in detail the following performance parameters: selectivity/specificity/identity, ruggedness/robustness, limit of detection, limit of quantification, decision limit and detection capability. With every method performance characteristic its essence and terminology are addressed, the current status of treating it is reviewed and recommendations are given, how to determine it, specifically in the case of LC–MS methods.     

Tutorial review on validation of liquid chromatography–mass spectrometry methods: Part II

This is the part II of a tutorial review intending to give an overview of the state of the art of method validation in liquid chromatography mass spectrometry (LC–MS) and discuss specific issues that arise with MS (and MS–MS) detection in LC (as opposed to the “conventional” detectors). The Part II starts with briefly introducing the main quantitation methods and then addresses the performance related to quantification: linearity of signal, sensitivity, precision, trueness, accuracy, stability and measurement uncertainty. The last section is devoted to practical considerations in validation. With every performance characteristic its essence and terminology are addressed, the current status of treating it is reviewed and recommendations are given, how to handle it, specifically in the case of LC–MS methods.     

A new reliable sample preparation for high throughput focused steroid profiling by gas chromatography–mass spectrometry

The use of steroid hormones as growth promoters in cattle has been banned within the European Union since 1988 but can still be fraudulently employed in animal breeding farms for anabolic purposes. If an efficient monitoring of synthetic compounds (screening and confirmation) is ensured today by many laboratories, pointing out suspicious samples from a natural steroids abuse remains a tricky challenge due to the difficulty to set relevant threshold levels for these endogenous compounds. The development of focused profiling or untargeted metabolomic approaches is then emerging in this context, with the objective to reveal potential biomarkers signing an exogenous administration of such natural steroids. This study aimed to assess sample preparation procedures based on microextraction and adapt them to high throughput urinary profiling or metabolomic analyses based on gas chromatography–mass spectrometry measurement. Two techniques have been tested and optimised, namely solid phase microextraction (SPME) and microextraction by packed sorbent (MEPS), using five model steroid metabolites (16α-hydroxyandrosterone, 2α-hydroxytestosterone, 11-keto,5β-androstanedione, 6α-hydroxyestradiol and 7β-hydroxypregnenolone). The considered performance criteria included not only the absolute response of the targeted compounds but also the robustness of the materials, and the global aspect of the diagnostic ion chromatograms obtained. After only five successive urinary extractions, a clear degradation of the SPME fiber was observed which led to discard this method as a relevant technique for profiling, whereas no degradation was observed on MEPS sorbent. Repeatability and recovery yields were calculated from urine samples fortified at 500 μg L⁻¹ and extracted by MEPS. They were found respectively below 11% and above 60% for all model compounds. Detection limits were in the 5–15 μg L⁻¹ range depending on the compounds, and a good linearity was observed on the 10–75 μg L⁻¹ range (R² > 0.99). This methodology was applied on urine samples collected from control versus androstenedione-treated bovines, revealing a significant concentration increase for several well-known metabolites such as etiocholanolone, 5α-androstane-3β,17α-diol, 5β-androstane-3α,17α-diol and 5-androstene-3β,17α-diol. Finally, these results allowed to confirm the suitability of the developed strategy and give to this new MEPS application a promising interest in the field of GC–MS based steroid profiling and metabolomic.     

Identification of polyethanolamines by chromatography–mass spectrometry

Presumable structures of polyethanolamines, synthesized by the catalytic β-hydroxyethylation of ammonia with an excess of ethylene oxide are determined by gas chromatography–electron ionization mass spectrometry and tandem mass spectrometry qualitatively, following fragmentation ways and also taking into account retention data. The preferable paths of consecutive reactions of the synthesis of polyethanolamines in a homologous series of isomers are found.     

Dried blood spots: Liquid chromatography–mass spectrometry analysis of Δ-tetrahydrocannabinol and its main metabolites

A sensitive and selective HPLC–MS/MS method has been developed for the first time for the analysis of Δ⁹-tetrahydrocannabinol (the most important active cannabinoid) and its hydroxylated and carboxylated metabolites in human Dried Blood Spots (DBSs). The simultaneous determination of Δ⁹-tetrahydrocannabinol and its two main metabolites allows assessing the time elapsed after the drug intake and distinguishing between acute or former consumption. This is an important information in specific contexts such as “on street” controls by police forces. DBSs have been chosen as the optimal biological matrix for this kind of testing, since they provide information on the actual state of intoxication, without storage and transportation problems usually associated with classical blood testing. The analysis is carried out on a C8 reversed phase column with a mobile phase composed of 0.1% formic acid in a water/methanol mixture and an electrospray ionisation (ESI) source, coupled to a triple quadrupole mass spectrometer. The method was validated according to international guidelines, with satisfactory results in terms of extraction yields, precision, stability and accuracy. Application to real DBS samples from Cannabis abusers gave reliable results, thus confirming the methodology suitability for roadside testing.     

Ultra-high-performance liquid chromatography–quadrupole time of flight tandem mass spectrometry based in vitro metabolite profiling of DK-GV-04P,a novel anticancer molecule under drug discovery

DK-GV-04P, chemically identified as 3-cinnamyl-2-(4-methoxyphenyl) quinazolin-4(3H)-one, is an investigational molecule synthesized at the Chemical Biology Laboratory of the National Institute of Pharmaceutical Education and Research-Ahmedabad. The compound has shown potential anticancer activity against squamous CAL27 cell lines. Metabolite identification and characterization are critical in drug discovery, providing key insights into a compound’s pharmacokinetics, pharmacodynamics safety, and metabolic fate. The primary aim of the study was to identify and characterize the in vitro metabolites of DK-GV-04P. In silico identification of the site of metabolism was also carried out using xenosite online software. The molecule was incubated with human liver microsomes and human S9 liver fraction to generate in vitro metabolites, which were further identified and characterized using ultra-high-performance liquid chromatography–quadrupole time of flight tandem mass spectrometry. A total of nine metabolites (four phase I and five phase II) were identified and characterized through tandem mass spectrometry. The major biotransformation pathways involved in metabolism of DK-GV-04P were hydroxylation, O-demethylation and glucuronidation. In addition to this, a detailed biotransformation pathway of DK-GV-04P has been established in this study.     

Liquid chromatography coordination ion-spray mass spectrometry (LC–CIS–MS) of docosahexaenoate ester hydroperoxides

Coordination ion-spray mass spectrometry (CIS–MS) is a useful tool in the detection and identification of complex mixtures of cholesterol ester and phospholipid hydroperoxides. The methyl ester, cholesterol ester, and phospholipid hydroperoxides of docosahexaenoic acid were analyzed by LC–CIS–MS and their elution orders were identified. Their corresponding alcohols were also identified. The methyl hydroperoxydocosahexaenoate (HPDHE) elution order is 14, 17, 16, 13, 20, 11, 10, 4, 7, 8 while the methyl hydroxydocosahexaenoate (HDHE) elution order is 14 17, 16, 13, 11, 10, 20, 7, 8, 4. The cholesteryl HPDHE elution order is 14, 17, 16, 13, 20, 11 10, 4, 7, 8 and the cholesteryl HDHE elution order is 17, 14, 16, 13, 11, 20, 10, 7, 8, 4. The elution order of the 1-palmitoyl-2-docosahexaenoyl-sn-glycero-3-phosphatidylcholine (PDPC) hydroperoxides and alcohols is 20, 16, 17, 13, 14, 10, 11, 7, 8, 4.Electronic supplementary material Supplementary material is available for this article if you access the article at . A link in the frame on the left on that page takes you directly to the supplementary material.     

Multi-residue off-flavour profiling in wine using stir bar sorptive extraction–thermal desorption–gas chromatography–mass spectrometry

A multi-residue method (MRM) for the detection and quantification of eight compounds responsible for off-flavours in wine using stir bar sorptive extraction (SBSE) followed by thermal desorption (TD) and gas chromatography–mass spectrometry (GC–MS) analysis is presented. The extraction and desorption conditions were optimised in order to get the best compromise for the simultaneous analysis of the eight target solutes, belonging to different chemical classes. The analytical conditions enable the quantification of the solutes below their respective organoleptic perception thresholds in wine. The method displayed good linearity over the concentration ranges explored in wine as well as excellent repeatability (RSD below 6%) and good reproducibility (RSD below 24%). The developed methodology was applied to the analysis of several wines and showed good agreement with the results collected with headspace solid-phase microextraction (HS-SPME) or liquid–liquid extraction (LLE) followed by GC–MS or electron capture detection (ECD). Good correlation was also found between the analytical and sensory results.     

Alkaloid profiling of the Chinese herbal medicine Fuzi by combination of matrix-assisted laser desorption ionization mass spectrometry with liquid chromatography–mass spectrometry

A matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) method was developed for the high throughput and robust qualitative profiling of alkaloids in Fuzi—the processed lateral roots of the Chinese herbal medicine Aconitum carmichaeli Debx (A. carmichaeli). After optimization, powdered roots – without any further sample preparation – could be used to screen for the presence of Aconitum alkaloids. Furthermore, the semi-quantitative potential of MALDI-MS was confirmed using liquid chromatography–mass spectrometry (LC–MS) as reference. In total over sixty alkaloids were detected by LC–MS and fifteen of them were tentatively identified. Both MALDI-MS and LC–MS analysis revealed significant variation in alkaloid content in different (commercial) samples. LC–MS analysis of three toxic alkaloids in 14 batches of Fuzi resulted in a variation of their concentrations expressed as RSDs of 138%, 99% and 221% for aconitine, hypaconitine and mesaconitine, respectively. The variation in concentrations (expressed as RSD) of about the ninety constituents detected were classified as follows: 13 constituents showed an RSD of 77–100%, 46 with an RSD of 100–150%, 21 with an RSD of 150–200% and 9 constituents with an RSD in concentration of 200–235%. These results demonstrate a strong difference in chemical composition of the various Fuzi and illustrate the necessity of adequate QA/QC procedures for both safety and efficiency of herbal medicine. The described analytical procedures for alkaloid profiling could play a role in these procedures.     

Study of archaeological artifact by chromatography–mass spectrometry

An archaeological artifact, representing a compound piece of the Bronze Age, the outer surface of which is encrusted with seeds, has been investigated. The chemical composition of the organic binder was determined by gas chromatography and mass spectrometry; X-ray fluorescence analysis was used to determine the nature of the inorganic base of the artifact.     

Recent advances on multidimensional liquid chromatography–mass spectrometry for proteomics: From qualitative to quantitative analysis—A review

With the acceleration of proteome research, increasing attention has been paid to multidimensional liquid chromatography–mass spectrometry (MDLC–MS) due to its high peak capacity and separation efficiency. Recently, many efforts have been put to improve MDLC-based strategies including “top-down” and “bottom-up” to enable highly sensitive qualitative and quantitative analysis of proteins, as well as accelerate the whole analytical procedure. Integrated platforms with combination of sample pretreatment, multidimensional separations and identification were also developed to achieve high throughput and sensitive detection of proteomes, facilitating highly accurate and reproducible quantification. This review summarized the recent advances of such techniques and their applications in qualitative and quantitative analysis of proteomes.