High-abundance peaks and peak clusters associate with pharmaceutical polymers and excipients in urinary untargeted clinical metabolomics data: exploration of their origin and possible impact on label-free quantification.

Pharmaceutical polymers and excipients represent interesting but often overlooked chemical classes in clinical exposure and bioanalytical research. These chemicals may cause hypersensitivity reactions, they can be useful to confirm exposure to pharmaceuticals, and they may pose bioanalytical challenges, including ion suppression in liquid chromatography-mass spectrometry (LC-MS-)based workflows. In this work, we assessed these chemicals in light of a rather surprising finding presented in two previously published studies, namely that usage of cyclosporine A, an immunosuppressive drug which is known to be cleared through excretion in the bile, explained the largest amount of variance in principal component analysis of urinary LC-SWATH/MS small-molecule profiling data. Specifically, we examined the freely-accessible 24-hour urine metabolomics data of 570 kidney transplant recipients included in the TransplantLines Biobank and Cohort Study (NCT03272841). These data unveiled thousands of high-abundance polymer peaks in some samples, which were associated with the use of the macrogol (i.e., polyethylene glycol) 3350 oral laxative agent. In addition, we found multiple clusters of high-abundance peaks which were linked to the exposure to two pharmaceutical excipients, namely short-chain polyethylene glycol (molecular weight <1000 Da) and polyethoxylated castor oil (also known as Kolliphor® EL or Cremophor® EL). Respectively, these excipients are used in temazepam capsules and cyclosporine A capsules, and the latter provides a plausible explanation for the rather surprising finding that instigated our work. Moreover, such explanation and our findings in general put emphasis on taking into consideration these and other pharmaceutical polymers and excipients when exploring, processing, and interpreting clinical small-molecule profiling data.

Structure-activity relationship studies and biological properties evaluation of peptidic NRP-1 ligands: Investigation of N-terminal cysteine importance.

Neuropilin-1 (NRP-1) is a major co-receptor of vascular endothelial growth factor receptor-2 (VEGFR-2). It may also stimulate tumour growth and metastasis independently of VEGF-A165. These functions make VEGF-A165/NRP-1 complex formation and its inhibition of great interest, where NRP-1 is the target for which effective ligands are sought. Design of peptide-like inhibitors represent a strategy with great potential in the treatment of NRP-1-related disorders. Here, we present the synthesis, molecular modelling, structure-activity relationship studies as well as biological evaluation of peptides with the branched sequences H2N-X-Lys(hArg)-Dab-Oic-Arg-OH and H2N-Lys(X-hArg)-Dab-Oic-Arg-OH. Two of the designed peptides, in which Cys was inserted in X position, expressed high affinity (∼40 nM value) for NRP-1 and were resistant to enzymatic digestion in human serum. Moreover, peptide/NRP-1 complex promoted fast intracytoplasmic protein trafficking towards the plasma membrane in breast cancer cells. Our results suggest that these compounds might be good candidates for further development of VEGF-A165/NRP-1 inhibitors.

Liquid chromatography and differential mobility spectrometry-data-independent mass spectrometry for comprehensive multidimensional separations in metabolomics.

The benefits of combining drift time ion mobility (DTIMS) with liquid chromatography-high-resolution mass spectrometry (HRMS) have been reported for metabolomics but the use of differential time mobility spectrometry (DMS) is less obvious due to the need for rapid scanning of the DMS cell. Drift DTIMS provides additional precursor ion selectivity and collisional cross-section information but the separation resolution between analytes remains cell- and component-dependent. With DMS, the addition of 2-propanol modifier can improve the selectivity but on cost of analyte MS response. In the present work, we investigate the liquid chromatography-mass spectrometry (LC-MS) analysis of a mix of 50 analytes, representative for urine and plasma metabolites, using scanning DMS with the single modifiers cyclohexane (Ch), toluene (Tol), acetonitrile (ACN), ethanol (EtOH), and 2-propanol (IPA), and a binary modifier mixture (cyclohexane/2-propanol) with emphasis on selectivity and signal sensitivity. 1.5% IPA in the N2 stream was found to suppress the signal of 50% of the analytes which could be partially recovered with the use of IPA to 0.05% as a Ch/IPA mixture. The potential to use the separation voltage/compensation voltage/modifier (SV/CoV/Mod) feature as an additional analyte identifier for qualitative analysis is also presented and applied to a data-independent LCxDMS-SWATH-MS workflow for the analysis of endogenous metabolites and drugs of abuse in human urine samples from traffic control.

Ultraviolet photodissociation and collision-induced dissociation for qualitative/quantitative analysis of low molecular weight compounds by liquid chromatography-mass spectrometry.

Collision-induced dissociation (CID) is the most wildly used fragmentation technique for qualitative and quantitative determination of low molecular weight compounds (LMWC). Ultraviolet photodissociation (UVPD) has been mainly investigated for the analysis of peptides and lipids while only in a limited way for LMWC. A triple quadrupole linear ion trap instrument has been modified to allow ultraviolet photodissociation (UVPD) in the end of the q2 region enabling various workflows with and without data-dependent acquisition (DDA) combining CID and UVPD in the same LC-MS analysis. The performance of UVPD, with a 266-nm laser, is compared to CID for a mix of 90 molecules from different classes of LMWC including peptides, pesticides, pharmaceuticals, metabolites, and drugs of abuse. These two activation methods offer complementary fragments as well as common fragments with similar sensitivities for most analytes investigated. The versatility of UVPD and CID is also demonstrated for quantitative analysis in human plasma of bosentan and its desmethyl metabolite, used as model analytes. Different background signals are observed for both fragmentation methods as well as unique fragments which opens the possibility of developing a selective quantitative assay with improved sample throughput, in particular for analytes present in different matrices.

Controlled Formation of Protonated and Radical Cation Precursor Ions by Atmospheric Pressure Photoionization with µLC-MS Enables Electron Ionization and MS/MS Library Search.

Atmospheric pressure photoionization (APPI) was developed as an alternative to electrospray ionization (ESI) for the generation of protonated molecules using liquid chromatography and optimized using dopants such as toluene, which predominantly forms protonated molecules, and chlorobenzene, which favors the formation of radical cations, although the latter has not been fully exploited. Based on 40 diverse low-molecular-weight compounds and micro liquid chromatography (µLC) coupled with APPI tandem mass spectrometry (APPI-MS/MS), the potential of radical cations was investigated. Chromatographic and ionization conditions were decoupled by post-column addition of methanol, allowing separate study and optimization. Due to the mass flow sensitive behavior of APPI, sensitivity is not affected by post-column dilution, and for 8 of 35 analytes, the radical cation response with µLC-APPI is better than for protonated molecules using µLC-ESI. Collision-induced fragmentation (CID) of radical cations produced within a collision energy range from 10-115 eV have, in the median, 65% of the fragments found in electron ionization (EI) spectra. This similarity allowed identification of 86% of the analytes using data-dependent acquisition (DDA) of radical cations and NIST EI library searches. We propose a workflow that uses multimodal DDA of protonated precursor molecules using ESI or APPI with toluene as a dopant, and radical cations produced by chlorobenzene-assisted µLC-APPI with post-column addition of methanol. This increases the confidence of molecular identification by allowing orthogonal library searches using MS/MS libraries for protonated precursor CID spectra and EI libraries for radical cation CID spectra.

Association of diuretic use with increased risk for long-term post-transplantation diabetes mellitus in kidney transplant recipients.

BACKGROUND: Post-transplantation diabetes mellitus (PTDM) is a major clinical problem in kidney transplant recipients (KTRs). Diuretic-induced hyperglycaemia and diabetes have been described in the general population. We aimed to investigate whether diuretics also increase PTDM risk in KTRs. METHODS: We included 486 stable outpatient KTRs (with a functioning graft ≥1 year) without diabetes from a prospective cohort study. Participants were classified as diuretic users and non-users based on their medication use verified by medical records. RESULTS: At the baseline study, 168 (35%) KTRs used a diuretic (thiazide, n = 74; loop diuretic, n = 76; others, n = 18) and 318 KTRs did not use a diuretic. After 5.2 years [interquartile range (IQR) 4.0‒5.9] of follow up, 54 (11%) KTRs developed PTDM. In Cox regression analyses, diuretic use was associated with incident PTDM, independent of age, sex, fasting plasma glucose (FPG) and haemoglobin A1c (HbA1c) {hazard ratio [HR] 3.28 [95% confidence interval (CI) 1.84-5.83]; P <0.001}. Further adjustment for potential confounders, including lifestyle, family history of cardiovascular disease, use of other medication, kidney function, transplantation-specific parameters, BMI, lipids and blood pressure did not materially change the association. Moreover, in Cox regression analyses, both thiazide and loop diuretics associated with the development of PTDM, independent of age, sex, FPG and HbA1c [HR 2.70 (95% CI 1.24-5.29); P = 0.012 and HR 5.08 (95% CI 2.49-10.34); P <0.001), respectively]. CONCLUSIONS: This study demonstrates that diuretics overall are associated with an increased risk of developing PTDM in KTRs, independent of established risk factors for PTDM development. The association was present for both thiazide and loop diuretics.

Analysis of illicit pills and drugs of abuse in urine samples using a 3D-printed open port probe hyphenated with differential mobility spectrometry-mass spectrometry.

The present work describes the application of an in-house developed 3D-printed open port probe (3DP-OPP) with differential ion mobility spectrometry (DMS) mass spectrometry. Targeted quantitative analysis in urine was performed with a triple quadrupole mass spectrometer in the selected reaction monitoring mode (OPP-DMS-SRM/MS) and illicit pill screening using data independent acquisition (OPP-DMS-SWATH/MS). The combination of compensation voltage (CoV) scanning in DMS using modifiers with SWATH/MS acquisition for MS/MS spectrum generation enabled the differentiation of isobaric signals with a large dynamic range and enhance the information contained in the screening of illicit ecstasy pills. As for any direct MS introduction technique where no chromatographic separation is applied DMS with acetonitrile as a modifier allows the separation of cocaine and tramadol, and their isomeric metabolites in urine samples. Quantitative application using OPP-DMS-SRM/MS is presented without the need for sample preparation with a lower limit of quantification at 10-25 ng mL-1 for the analytes and less than 40 seconds for sample to sample analysis.

Mass spectrometry based high-throughput bioanalysis of low molecular weight compounds: are we ready to support personalized medicine?

Liquid chromatography coupled to mass spectrometry (LC-MS) is the gold standard in bioanalysis for the development of quantitative assays to support drug development or therapeutic drug monitoring. High-throughput and low-cost gene sequencing have enabled a paradigm shift from one treatment fits all to personalized medicine (PM). However, gene monitoring provides only partial information about the health state. The full picture requires the combination of gene monitoring with the screening of exogenous compounds, metabolites, lipids, and proteins. This critical review discusses how mass spectrometry-based technologies and approaches including separation sciences, ambient ionization, and ion mobility are/could be used to support high-throughput bioanalysis of endogenous end exogenous low molecular weight compounds. It includes also various biological sample types (from blood to expired air), and various sample preparation techniques.

Effects of the LC mobile phase in vacuum differential mobility spectrometry-mass spectrometry for the selective analysis of antidepressant drugs in human plasma.

The effect of LC mobile phase composition and flow rate (2-50 µL/min) on mobility behavior in vacuum differential mobility spectrometry (vDMS) was investigated for electrosprayed isobaric antidepressant drugs (AD); amitriptyline, maprotiline, venlafaxine; and structurally related antidepressants nortriptyline, imipramine, and desipramine. While at 2 µL/min, no difference in compensation voltage was observed with methanol and acetonitrile, at 50 µL/min, acetonitrile used for LC elution of analytes enabled the selectivity of the mobility separation to be improved. An accurate and sensitive method could be developed for the quantification of six AD drugs in human plasma using trap/elute micro-LC setup hyphenated to vDMS with mass spectrometric detection in the selected ion monitoring mode. The assay was found to be linear over three orders of magnitude, and the limit of quantification was of 25 ng/mL for all analytes. The LC-vDMS-SIM/MS method was compared to a LC-MRM/MS method, and in both cases, inter-assay precisions were lower than 12.5 and accuracies were in the range 91.5-110%, but with a four times reduced analysis time (2 min) for the LC-vDMS-SIM/MS method. This work illustrates that with vDMS, the LC mobile phase composition can be used to tune the ion mobility separation and to improve assay selectivity without additional hardware.

Clustering and Nonclustering Modifier Mixtures in Differential Mobility Spectrometry for Multidimensional Liquid Chromatography Ion Mobility-Mass Spectrometry Analysis.

Modifiers provide fast and reliable tuning of separation in differential mobility spectrometry (DMS). DMS selectivity for separating isomeric molecules depends on the clustering modifier concentration, which is typically 1.5-3 mol % ratio of isopropanol or ethanol in nitrogen. Low concentrations (0.1%) of isopropanol were found to improve resolution and sensitivity but at the cost of practicality and robustness. Replacing the single-channel DMS pump with a binary high-performance liquid chromatography (HPLC) pump enabled the generation of modifier mixtures at a constant flow rate using an isocratic or gradient mode, and the analytical benefits of the system were investigated considering cyclohexane, n-hexane, or n-octane as nonclustering modifiers and isopropanol or ethanol as clustering modifiers. It was found that clustering and nonclustering modifier mixtures enable optimization of selectivity, resolution, and sensitivity for different positional isomers and diastereoisomers. Data further suggested different ion separation mechanisms depending on the modifier ratios. For 85 analytes, the absolute difference in compensation voltages (CoVs) between pure nitrogen and cyclohexane at 1.5 mol % ratio was below 4 V, demonstrating its potential as a nonclustering modifier. Cyclohexane's nonclustering behavior was further supported by molecular modeling using density functional theory (DFT) and calculated cluster binding energies, showing positive ΔG values. The ability to control analyte CoVs by adjusting modifier concentrations in isocratic and gradient modes is beneficial for optimizing multidimensional LCxDMS-MS. It is fast and effective for manipulating the DMS scanning window size to realize shorter mass spectrometry (MS) acquisition cycle times while maintaining a sufficient number of CoV steps and without compromising DMS separation performance.

Adduct annotation in liquid chromatography/high-resolution mass spectrometry to enhance compound identification.

Annotation and interpretation of full scan electrospray mass spectra of metabolites is complicated by the presence of a wide variety of ions. Not only protonated, deprotonated, and neutral loss ions but also sodium, potassium, and ammonium adducts as well as oligomers are frequently observed. This diversity challenges automatic annotation and is often poorly addressed by current annotation tools. In many cases, annotation is integrated in metabolomics workflows and is based on specific chromatographic peak-picking tools. We introduce mzAdan, a nonchromatography-based multipurpose standalone application that was developed for the annotation and exploration of convolved high-resolution ESI-MS spectra. The tool annotates single or multiple accurate mass spectra using a customizable adduct annotation list and outputs a list of [M+H]+ candidates. MzAdan was first tested with a collection of 408 analytes acquired with flow injection analysis. This resulted in 402 correct [M+H]+ identifications and, with combinations of sodium, ammonium, and potassium adducts and water and ammonia losses within a tolerance of 10 mmu, explained close to 50% of the total ion current. False positives were monitored with mass accuracy and bias as well as chromatographic behavior which led to the identification of adducts with calcium instead of the expected potassium. MzAdan was then integrated in a workflow with XCMS for the untargeted LC-MS data analysis of a 52 metabolite standard mix and a human urine sample. The results were benchmarked against three other annotation tools, CAMERA, findMAIN, and CliqueMS: findMAIN and mzAdan consistently produced higher numbers of [M+H]+ candidates compared with CliqueMS and CAMERA, especially with co-eluting metabolites. Detection of low-intensity ions and correct grouping were found to be essential for annotation performance. Graphical abstract.

Mass spectrometry based approaches and strategies in bioanalysis for qualitative and quantitative analysis of pharmaceutically relevant molecules.

Mass spectrometry plays an essential role in qualitative and quantitative analysis of pharmaceutically relevant molecules. The present review summarizes some the most common applications of LC-MS for the characterization of therapeutic low-molecular-weight compounds, peptides and proteins, and oligonucleotides using low-resolution and high-resolution tandem mass spectrometry. In addition, the benefit of multistage MS, differential ion mobility, and data independent acquisition is emphasized. At last, the potential of coupling MS with novel interfaces for high-throughput analysis is discussed.

SWATH-MS for metabolomics and lipidomics: critical aspects of qualitative and quantitative analysis.

INTRODUCTION: While liquid chromatography coupled to mass spectrometric detection in the selected reaction monitoring detection mode offers the best quantification sensitivity for omics, the number of target analytes is limited, must be predefined and specific methods developed. Data independent acquisition (DIA), including SWATH using quadrupole time of flight or orbitrap mass spectrometers and generic acquisition methods, has emerged as a powerful alternative technique for quantitative and qualitative analyses since it can cover a wide range of analytes without predefinition. OBJECTIVES: Here we review the current state of DIA, SWATH-MS and highlight novel acquisition strategies for metabolomics and lipidomics and opportunities for data analysis tools. METHOD: Different databases were searched for papers that report developments and applications of DIA and in particular SWATH-MS in metabolomics and lipidomics. RESULTS: DIA methods generate digital sample records that can be mined retrospectively as further knowledge is gained and, with standardized acquisition schemes, used in multiple studies. The different chemical spaces of metabolites and lipids require different specificities, hence different acquisition and data processing approaches must be considered for their analysis. CONCLUSIONS: Although the hardware and acquisition modes are well defined for SWATH-MS, a major challenge for routine use remains the lack of appropriate software tools capable of handling large datasets and large numbers of analytes.

Hyphenation of packed column supercritical fluid chromatography with mass spectrometry: where are we and what are the remaining challenges?

Over the last decades, packed column supercritical fluid chromatography (pSFC) using carbon dioxide (CO2) as supercritical fluid has gained interest as a complementary separation technique to liquid chromatography (LC). Various commercial solutions for the hyphenation to atmospheric pressure ionization (API) including electrospray (ESI) and atmospheric pressure chemical ionization (APCI) have been described using split-flow or full-flow introduction approaches. This review discusses various aspects of the hyphenation using these two approaches. It also illustrates the difference between LC-MS and SFC-MS. The benefits and challenges of the decoupling of the separation mobile phase from the ionization conditions are also pointed out. The effect of CO2 on ESI performance and the adduct reduction are also discussed. Finally, limitation of current hardware and the limited use of smaller column internal diameters (i.d.) are discussed. Graphical abstract.

Urea-Peptide Hybrids as VEGF-A165/NRP-1 Complex Inhibitors with Improved Receptor Affinity and Biological Properties.

Neuropilin-1 (NRP-1), the major co-receptor of vascular endothelial growth factor receptor-2 (VEGFR-2), may also independently act with VEGF-A165 to stimulate tumour growth and metastasis. Therefore, there is great interest in compounds that can block VEGF-A165/NRP-1 interaction. Peptidomimetic type inhibitors represent a promising strategy in the treatment of NRP-1-related disorders. Here, we present the synthesis, affinity, enzymatic stability, molecular modeling and in vitro binding evaluation of the branched urea-peptide hybrids, based on our previously reported Lys(hArg)-Dab-Oic-Arg active sequence, where the Lys(hArg) branching has been modified by introducing urea units to replace the peptide bond at various positions. One of the resulting hybrids increased the affinity of the compound for NRP-1 more than 10-fold, while simultaneously improving resistance for proteolytic stability in serum. In addition, ligand binding to NRP-1 induced rapid protein stock exocytotic trafficking to the plasma membrane in breast cancer cells. Examined properties characterize this compound as a good candidate for further development of VEGF165/NRP-1 inhibitors.

Loss of NifQ Leads to Accumulation of Porphyrins and Altered Metal-Homeostasis in Nitrogen-Fixing Symbioses.

Symbiotic nitrogen fixation between legumes and rhizobia involves a coordinated expression of many plant and bacterial genes as well as finely tuned metabolic activities of micro- and macrosymbionts. In spite of such complex interactions, symbiotic proficiency remains a resilient process, with host plants apparently capable of compensating for some deficiencies in rhizobia. What controls nodule homeostasis is still poorly understood and probably varies between plant species. In this respect, the promiscuous Sinorhizobium (Ensifer) fredii strain NGR234 has become a model to assess the relative contribution of single gene products to many symbioses. Here, we describe how a deletion in nifQ of NGR234 (strain NGRΔnifQ) makes nodules of Vigna unguiculata, V. radiata, and Macroptilium atropurpureum but not of the mimisoid tree Leucaena leucocephala, purple-red. This peculiar dark-nodule phenotype did not necessarily correlate with a decreased proficiency of NGRΔnifQ but coincided with a 20-fold or more accumulation of coproporphyrin III and uroporphyrin III in V. unguiculata nodules. Porphyrin accumulation was not restricted to plant cells infected with bacteroids but also extended to the nodule cortex. Nodule metal-homeostasis was altered but not sufficiently to prevent assembly and functioning of nitrogenase. Although the role of NifQ in donating molybdenum during assembly of nitrogenase cofactor FeMo-co makes it essential in free-living diazotrophs, our results highlight the dispensability of NifQ in many legume species.

Modifier Selectivity Effect on Differential Ion Mobility Resolution of Isomeric Drugs and Multidimensional Liquid Chromatography Ion Mobility Analysis.

Cluster formation in the alternating electric field during differential ion mobility is critical for separation selectivity and is governed by two factors. One is the reduced mass, and the other factor is cluster binding energy between an ion and a neutral solvent molecule (modifier). Therefore, separations of isomeric analytes using a modifier can be related to the thermochemistry of the cluster formation, as subtle changes in the molecular structure will affect its charge delocalization and the binding energy with the corresponding modifier will be different. We have examined the relationship between calculated Gibbs free energies of the cluster formation and experimental ion mobility measurements (CoV dispersion plots) considering the most prominent ion-modifier interactions: charge-dipole, dipole-dipole, and charge-quadrupole. To explain selectivity effects due to the modifier, we have selected a series of positional isomers of sulfonamide drugs that were analyzed in positive and negative electrospray and the diastereoisomers ephedrine and pseudoephedrine in positive mode. The following modifiers were investigated: water, linear and branched alcohols, acetonitrile, acetone, toluene, and ethyl acetate. We could demonstrate a dependence of the separation selectivity of the differential mobility on the reduced mass and Gibbs free energy of the cluster formation. These results are supported by thermochemistry calculations (DFT) and interpreted by molecular modeling. Finally, we describe differential mobility spectrometry selectivity tuning for the multidimensional LCxDMS-MS separation of sulfonamide isomers in human plasma.

Urea moiety as amide bond mimetic in peptide-like inhibitors of VEGF-A165/NRP-1 complex.

NRP-1 is an important co-receptor of vascular endothelial growth factor receptor-2 (VEGFR-2). Many reports suggested that NRP-1 might also serve as a separate receptor for VEGF-A165 causing stimulation of tumour growth and metastasis. Therefore, compounds interfering with VEGF-A165/NRP-1 complex triggered interest in the design of new molecules, including peptides, as anti-angiogenic and anti-tumour drugs. Here, we report the synthesis, affinity and stability evaluation of the urea-peptide hybrids, based on general Lys(hArg)-AA2-AA3-Arg sequence, where hArg residue was substituted by Arg urea unit. Such substitution does not substantially affected affinity of compounds for NRP-1 but significantly increased their proteolytic stability in plasma.

Hybrid SWATH/MS and HR-SRM/MS acquisition for phospholipidomics using QUAL/QUANT data processing.

A hybrid SWATH/MS and HR-SRM/MS acquisition approach using multiple unit mass windows and 100 u precursor selection windows has been developed to interface with a chromatographic lipid class separation. The method allows for the simultaneous monitoring of sum compositions in MS1 and up to 48 lipids in MS2 per lipid class. A total of 240 lipid sum compositions from five phospholipid classes could be monitored in MS2 (HR-SRM/MS) while there was no limitation in the number of analytes in MS1 (HR-SIM/MS). On average, 92 lipid sum compositions and 75 lipid species could be quantified in human plasma samples. The robustness and precision of the workflow has been assessed using technical triplicates of the subject samples. Lipid identification was improved using a combined qualitative and quantitative data processing based on prediction instead of library search. Lipid class specific extracted ion currents of precursors and the corresponding molecular species fragments were extracted based on the information obtained from lipid building blocks and a combinatorial strategy. The SWATH/MS approach with the post-acquisition processing is not limited to the analyzed phospholipid classes and can be applied to other analytes and samples of interest. Graphical abstract.