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Infrared ion spectroscopy is increasingly recognized as a method to identify mass spectrometry-detected analytes in many (bio)chemical areas and its integration in analytical laboratories is now on the horizon. Commercially available quadrupole ion trap mass spectrometers are attractive ion spectroscopy platforms but operate at relatively high pressures. This promotes collisional deactivation which directly interferes with the multiple-photon excitation process required for ion spectroscopy. To overcome this, infrared lasers having a high instantaneous power are required and therefore a majority of analytical studies have been performed at infrared free electron laser facilities. Proliferation of the technique to routine use in analytical laboratories requires table-top infrared lasers and optical parametric oscillators (OPOs) are the most suitable candidates, offering both relatively high intensities and reasonable spectral tuning ranges. Here, we explore the potential of a range of commercially available high-power OPOs for ion spectroscopy, comparing systems with repetition rates of 10 Hz, 20 kHz, 80 MHz and a continuous-wave (cw) system. We compare the performance for various molecular ions and show that the kHz and MHz repetition-rate systems outperform cw and 10 Hz systems in photodissociation efficiency and offer several advantages in terms of cost-effectiveness and practical implementation in an analytical laboratory not specialized in laser spectroscopy., Evaluation of four table-top IR lasers for ion spectroscopy in ion trap mass spectrometers shows high rep-rate lasers offer better photodissociation efficiency and are more cost-effective and practical compared to low rep-rate or cw alternatives.

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The identification of biotransformation products of drug compounds is a crucial step in drug development. Over the last decades, liquid chromatography-mass spectrometry (LC-MS) has become the method of choice for metabolite profiling because of its high sensitivity and selectivity. However, determining the full molecular structure of the detected metabolites, including the exact biotransformation site, remains challenging on the basis of MS alone. Here we explore infrared ion spectroscopy (IRIS) as a novel MS-based method for the elucidation of metabolic pathways in drug metabolism research. Using the drug midazolam as an example, we identify several biotransformation products directly from an in vitro drug incubation sample. We show that IR spectra of the aglycone MS/MS fragment ions of glucuronide metabolites establish a direct link between detected phase I and phase II metabolites. Moreover, using quantum-chemically computed IR spectra of candidate structures, we are able to assign the exact sites of biotransformation in absence of reference standards. Additionally, we demonstrate the utility of IRIS for structural elucidation by identifying several ring-opened midazolam derivatives formed in an acidic environment.

This study reports on the potential of using ion-exchange suppressor technology in liquid chromatography—electrospray ionization mass spectrometry workflows. The aim was to use high salt concentrations to improve separation performance, while overcoming the resulting significant ion suppression during electrospray ionization. As a case study, we apply suppressor technology to the hydrophilic interaction liquid chromatography separation and detection of taurine and glycochenodeoxycholate sulfate, endogenous biomarkers for organic anion transporter protein inhibition. The desired chromatographic selectivity was achieved applying 100 mM ion-pairing agent, while competing ions negatively affecting MS sensitivity were actively removed post-column from the solvent via a charged partially permeable membrane and replaced with protons, resulting in an up to 10-fold increase in detection sensitivity.

This article describes in vivo biotransformation and disposition of erdafitinib following single oral dose of 3H-erdafitinib and 14C-erdafitinib to intact and bile duct-cannulated (BC) rats (4 mg/k...

The human absorption, distribution, metabolism, and excretion (hADME) study is the cornerstone of the clinical pharmacology package for small molecule drugs, providing comprehensive information on the rates and routes of disposition and elimination of drug-related material in humans through the use of

The neglected tropical disease onchocerciasis, or river blindness, is caused by infection with the filarial nematode Onchocerca volvulus. Current estimates indicate that 17 million people are infected worldwide, the majority of them living in Africa. Today there are no non-invasive tests available that can detect ongoing infection, and that can be used for effective monitoring of elimination programs. In addition, to enable pharmacodynamic studies with novel macrofilaricide drug candidates, surrogate endpoints and efficacy biomarkers are needed but are non-existent. We describe the use of a multimodal untargeted mass spectrometry-based approach (metabolomics and lipidomics) to identify onchocerciasis-associated metabolites in urine and plasma, and of specific lipid features in plasma of infected individuals (O. volvulus infected cases: 68 individuals with palpable nodules; lymphatic filariasis cases: 8 individuals; non-endemic controls: 20 individuals). This work resulted in the identification of elevated concentrations of the plasma metabolites inosine and hypoxanthine as biomarkers for filarial infection, and of the urine metabolite cis-cinnamoylglycine (CCG) as biomarker for O. volvulus. During the targeted validation study, metabolite-specific cutoffs were determined (inosine: 34.2 ng/ml; hypoxanthine: 1380 ng/ml; CCG: 29.7 ng/ml) and sensitivity and specificity profiles were established. Subsequent evaluation of these biomarkers in a non-endemic population from a different geographical region invalidated the urine metabolite CCG as biomarker for O. volvulus. The plasma metabolites inosine and hypoxanthine were confirmed as biomarkers for filarial infection. With the availability of targeted LC-MS procedures, the full potential of these 2 biomarkers in macrofilaricide clinical trials, MDA efficacy surveys, and epidemiological transmission studies can be investigated., Author summary Today’s diagnosis of infection with the filarial parasite Onchocerca volvulus mainly depends on the microscopic analysis of skin biopsies and serological testing. The work presented here describes the use of multiple mass spectrometry-based screening methods (metabolomics and lipidomics) to search for biomarkers indicative of infection with Onchocerca volvulus. This resulted in the identification of elevated concentrations of the plasma metabolites inosine and hypoxanthine as biomarkers for filarial infection, and of the urine metabolite cis-cinnamoylglycine as biomarker for O. volvulus. Further evaluation of these biomarkers in a geographically distinct non-endemic population however invalidated the use of urine cis-cinnamoylglycine. These findings are of utmost importance as it not only opens new avenues in the development of non-invasive diagnostic tools for filarial infections, but also emphasizes the need for evaluation and validation of newly discovered biomarkers in different populations from different geographies.

Distinguishing positional isomers, such as compounds having different substitution patterns on an aromatic ring, presents a significant challenge for mass spectrometric analyses and is a frequently encountered difficulty in, for example, drug metabolism research. Here, we demonstrate infrared ion spectroscopy (IRIS) as a promising new mass spectrometry-based technique that easily differentiates between positional isomers of disubstituted phenyl-containing compounds. By analyzing different substitution patterns over several sets of isomeric compounds, we show that IRIS produces a highly consistent and distinct pattern of IR bands, especially in the range between 650 and 900 cm-1, that are mostly independent of the specific chemical functionality contained in the substituent group. These patterns are accurately predicted by quantum-chemically computed IR spectra and correspond well with tabulated IR group-frequencies known from conventional absorption spectroscopy. Therefore, we foresee that this method will be generally applicable to disubstituted phenyl-containing compounds and that direct interpretation of experimental IRIS spectra in terms of ortho-, meta- or para-substitution is possible, even without comparison to experimental or computationally predicted reference spectra. Strategies for the analysis of larger compounds having more congested IR spectra as well as of compounds having low (electrospray) ionization efficiencies are presented in order to demonstrate the broad applicability of this methodology.

In this phase 1 study, the absolute bioavailability and absorption, metabolism, and excretion (AME) of apalutamide, a competitive inhibitor of the androgen receptor, were evaluated in 12 healthy men. Subjects received 240 mg of apalutamide orally plus a 15-minute intravenous infusion of 100 µg of apalutamide containing 9.25 kBq (250 nCi) of 14C-apalutamide (2 hours postdose) for absolute bioavailability assessment or plus one 400-µg capsule containing 37 kBq (1000 nCi) of 14C-apalutamide for AME assessment. Content of 14C and metabolite profiling for whole blood, plasma, urine, feces, and expired air samples were analyzed using accelerator mass spectrometry. Apalutamide absolute oral bioavailability was ≈100%. After oral administration, apalutamide, its N-desmethyl metabolite (M3), and an inactive carboxylic acid metabolite (M4) accounted for most 14C in plasma (45%, 44%, and 3%, respectively). Apalutamide elimination was slow, with a mean plasma half-life of 151-178 hours. The mean cumulative recovery of total 14C over 70 days postdose was 64.6% in urine and 24.3% in feces. The urinary excretion of apalutamide, M3, and M4 was 1.2%, 2.7%, and 31.1% of dose, respectively. Fecal excretion of apalutamide, M3, and M4 was 1.5%, 2.0%, and 2.4% of dose, respectively. Seventeen apalutamide metabolites and six main metabolic clearance pathways were identified. In vitro studies confirmed CYP2C8 and CYP3A4 roles in apalutamide metabolism.

Local delivery to the lower gut to treat diseases of the colon has become a topic of special attention. Tissue exposure of locally acting agents is not represented by plasma concentrations. Therefore, reliable methods to measure tissue uptake at the primary site of action (e.g., epithelial layer or lamina propria) are vital. This work investigates the suitability of mass spectrometry imaging (MSI) in quantitatively visualizing intestinal transmural drug distribution. Tofacitinib (Tofa), a drug approved for the treatment of several autoimmune diseases, including ulcerative colitis, was selected as a tool compound for feasibility studies. Oneand 7-h postdose sections of the ileum, proximal- and distal-colon from rats that received an oral solution of Tofa were subjected to matrix-assisted laser desorption ionization (MALDI)-MSI. A dilution series of individual concentrations sprayed over an entire tissue section allowed for tissue type-specific quantitation. At 1 h (systemic T-max), the signal was highest in the ileum, whereas at 7 h, the signal was highest in the colon, when the unabsorbed fraction of the compound reached the colon. A combination of three-dimensional (3D) intensity plots and hematoxylin and eosin (H&E) stains showed a visually observable gradual decrease in Tofa concentration from the lumen toward the muscular layer of the proximal colon. The high luminal concentration of Tofa indicated that flushing of the intestines with saline does not result in complete removal of the drug material from the lumen. This could cause an overestimation of drug concentration in gut tissue homogenates by conventional liquid chromatography-mass spectrometry (LC-MS) methods. This study demonstrates the utility of MSI to differentiate between the lumen and intestinal wall layers and enables proper interpretation of tissue distribution data.

This study aimed to systematically explore compositional effects for a series of lipid systems, on the in vitro drug solubilization and in vivo bioavailability of three poorly water-soluble drugs with different physico-chemical properties. While many lipid-based drug products have successfully reached the market, there is still a level of uncertainty on the design guidelines for such drug products with limited understanding on the influence of composition on in vitro and in vivo performance. Lipid-based drug delivery systems were prepared using either single excipient systems based on partially digested triglycerides (i.e. mono- and/or di-glycerides) or increasingly complex systems by incorporating surfactants and/or triglycerides. These lipid systems were evaluated for both in vitro and in vivo behaviour. Results indicated that simple single component long chain lipid systems are more beneficial for the absorption of the weak acid celecoxib and the weak base cinnarizine compared to equivalent single component medium chain lipid systems. Similarly, a two-component system produced by incorporating small amount of hydrophilic surfactant yields similar overall pharmacokinetic effects. The lipid drug delivery systems based on medium chain lipid excipients improved the in vivo exposure of the neutral drug JNJ-2A. The higher in vivo bioavailability of long chain lipid systems compared to medium chain lipid systems was in agreement with in vitro dilution and dispersion studies for celecoxib and cinnarizine. The present study demonstrated the benefits of using mono-/di-glycerides as single component excipients in LBDDS to streamline formulation screening and improve oral bioavailability for the three tested poorly water-soluble drugs.

Rationale In liquid chromatography/mass spectrometry (LC/MS) the LC flow is often split prior to the mass spectrometer, for instance, when collecting fractions of the separated sample for other purposes or when less sensitive parallel detection is applied. The aim of this study is to optimize the actual split ratio and make-up flow composition. Methods Different types of splitters were evaluated in combination with a make-up flow. A home-made 1/10 T-piece splitter and commercial 1/10, 1/100 and 1/250 splitters were evaluated by continuous and accurate measurements of the actual split ratio throughout the LC gradient. The make-up flow composition was optimized for maximum electrospray ionization (ESI)-MS sensitivity in the positive mode using ESI efficiency measurements. Results Altogether 22 different solvent conditions were tested on 20 pharmaceutical compounds with a wide variety of functional groups and physicochemical properties (molecular weight, logP, and pKa ). Methanol/10 mM formic acid in water (90/10) provided on average the best results. Conclusions Methanol/10 mM formic acid in water (90/10) proved to be the best make-up flow composition in relation to the average sensitivity obtained. Stronger acidic conditions using oxalic acid or higher formic acid concentrations had a clear positive effect on the sensitivity of compounds with low ionization efficiency. The tested split ratios were relatively stable over the main part of the gradient but showed some variation at very low and very high organic conditions. Differences were larger with methanol compared with acetonitrile containing solvent compositions and when applied without a column or with very long connecting tubing.

Chemical derivatization allows the introduction of an “ICP-MS accessible” element into pharmaceuticals and their metabolites, thus extending the application range of HPLC-ICP-MS in pharmaceutical applications. In this study, such a pre-column derivatization approach was developed with the aim of using HPLC-ICP-MS for the quantitative metabolite profiling of pharmaceutical drugs in human plasma samples. We targeted the amino and hydroxyl groups, two of the most abundant functional groups present in drugs, and used 4-aminopyridine and 3-hydroxy-4-aminopyridine as model compounds. 4-Iodobenzoyl chloride was used as a commercially available and multi-functional derivatization reagent to introduce iodine (I) as an element that can be quantified using ICP-MS with high sensitivity. This reagent reacts readily with the amino group present in 4-aminopyridine and the amino and hydroxyl groups present in its metabolite 3-hydroxy-4-aminopyridine. The reaction conditions were systematically optimized to guarantee quantitative formation of the target derivatives. Baseline separation of the target derivatives from one another and from also derivatized endogenous compounds was achieved using reversed phase UHPLC and gradient elution. Accuracy (recovery between 95 and 107%) and precision (repeatability ≤ 6.9% RSD) were fit-for-purpose for both 4-aminopyridine and 3-hydroxy-4-aminopyridine. The limit of quantification (LOQ) is 10 μg L−1 for I, corresponding to 7.5 μg L−1 of 4-aminopyridine and 5.8 μg L−1 of 3-hydroxy-4-aminopyridine, respectively.

High-resolution mass spectrometry (HRMS) in full scan mode acquires all ions present in the sample of interest offering a lot of qualitative information. This, in combination with the improved performance of the new generation HRMS systems, triggers more (bio) analysts to switch from triple quad MS systems to HRMS for quantitative analysis. Quantitative processing of HRMS data is performed based on narrow mass extraction windows rather than on nominal mass product ion chromatograms (SRM or MRM). Optimal processing of HRMS data requires different considerations and software tools and can have an impact on data processing and final results. The selection of centroid versus continuum/profile data for processing, selection of the optimal narrow mass extraction window, using theoretical versus measured accurate mass for the extraction of the ion chromatograms as well as differences in calculations and data handling residing in the different vendor software packages are tackled in the presented manuscript. These differences are illustrated on HRMS data acquired for the same plasma samples on three different platforms, i.e., a Sciex QToF, a Waters QToF, and a Thermo Orbitrap system, and processed in four different software packages, i.e., Sciex Analyst® TF, Waters Masslynx, Waters Unifi, and Thermo Xcalibur. The impact of these differences on quantitative HRMS performance was evaluated on calibration curves of eight small molecule compounds in plasma using four different ways of processing. Simple guidelines are provided for the selection of an optimal mass extraction window for continuum and centroided data. Graphical Abstract.

The importance of metabolites is assessed based on their abundance. Most of the metabolites are at present identified based on ESI/MS measurements and the relative abundance is assessed from the relative peak areas of these metabolites. Unfortunately, relative intensities can be highly misleading as different compounds ionise with vastly different efficiency in the ESI source and matrix components may cause severe ionisation suppression. In order to reduce this inaccuracy, we propose predicting the ionisation efficiencies of the analytes in seven biological matrices (neat solvent, blood, plasma, urine, cerebrospinal fluid, brain and liver tissue homogenates). We demonstrate, that this approach may lead to an order of magnitude increase in accuracy even in complicated matrices. For the analyses of 10 compounds, mostly drugs, in negative electrospray ionisation mode we reduce the predicted abundance mismatch compared to the actual abundance on average from 660 to 8 times. The ionisation efficiencies were predicted based on i) the charge delocalisation parameterWAPS and ii) the degree of ionisation a, and the prediction model was subsequently validated based on the cross-validation method 'leave-one-out'. (C) 2018 Elsevier B.V. All rights reserved.

Drug Metabolism and Pharmacokinetics (DMPK) is a core scientific discipline within drug discovery and development as well as post-marketing. It helps to design and select the most promising drug candidate and obtain advanced insights on the processes that control absorption, distribution, metabolism and excretion (ADME) of the final drug candidate. Mass spectrometry is one of the key technologies applied in DMPK. Therefore, the continuous advances made in the field of mass spectrometry also directly impact the way in which we investigate the ADME properties of a compound, providing us with new tools to gather more information or improve our efficiency. An overview will be given of some important current trends and future perspectives in the field.

The intestinal barrier controls intestinal permeability, and its disruption has been associated with multiple diseases. Therefore, preclinical safety biomarkers monitoring barrier integrity are essential during the development of drugs targeting the intestines, particularly if starting treatment early after onset of disease. Classical toxicology endpoints are not sensitive enough and therefore our objective was to identify non-invasive markers enabling early in vivo detection of colonic barrier perturbation. Male Sprague-Dawley rats were dosed intracolonically via the rectum, using sodium caprate or ibuprofen as tool compounds to alter barrier integrity. Several potentially translational biomarkers and probe molecules related to permeability, inflammation or tissue damage were evaluated, using various analytical platforms, including immunoassays, targeted metabolomics and highly sensitive ultra-performance liquid chromatography-tandem mass spectrometry. Several markers were identified that allow early in vivo detection of colonic barrier integrity changes, before histopathological evidence of tissue damage. The most promising permeability markers identified were plasma fluorescein isothiocyanate-dextran 4000 and a lactulose/mannitol/sucralose mixture in urine. These markers showed maximum increases over 100-fold or approximately 10-50-fold, respectively. Intracolonic administration of the above probe molecules outperformed oral administration and inflammatory or other biomarkers, such as α2 -macroglobulin, calprotectin, cytokines, prostaglandins and a panel of metabolic molecules to identify early and subtle changes in barrier integrity. However, optimal timing of probe administration and sample collection is important for all markers evaluated. Inclusion of these probe molecules in preclinical toxicity studies might aid in risk assessment and the design of a clinical biomarker plan, as several of these markers have translational potential.

The development of suitable analytical methods for drug ADME (absorption, distribution, metabolism and excretion) studies is of great importance. The currently routinely applied detection techniques usually demonstrate a structure-dependent analytical response (MS-based method) or require the synthesis of a radiolabelled version of the parent drug (radiodetection) for accurate quantification. Inductively coupled plasma-(tandem) mass spectrometry (ICP-MS(/MS)) offers a promising alternative to radiolabelling followed by radiodetection due to the structure-independent nature of its analytical response. Within the context of this study, an accurate, simple and sensitive HPLC-ICP-MS/MS method for the quantitative metabolite profiling of diclofenac in human plasma based on the pre-column derivatization of the carboxylic and phenolic –OH groups present in the parent drug and its major metabolite, 4′-hydroxy-diclofenac, was developed and validated. A cost-effective and commercially available derivatization reagent, p-bromophenacyl bromide (p-BPB), was applied for the introduction of Br into the drug molecule and its major metabolite, enabling the element-selective detection and quantification based on the Br-signal. The presence of Cl in both diclofenac and 4′-hydroxy-diclofenac allowed an additional validation via simultaneous monitoring of the Cl-signal by using a state-of-art ICP-MS/MS instrument equipped with a collision/reaction cell. The reaction conditions were successfully optimized to achieve a quantitative formation of the corresponding derivatization products, while the baseline separation of the target compounds in a typical biological matrix (i.e. human plasma) was achieved using gradient reversed phase high-performance liquid chromatography (RP-HPLC). A fit-for-purpose accuracy (recovery between 85–115%) and precision (repeatability ≤7.2% RSD) were achieved. The limits of quantification (LOQ) are ≈50 μg L−1 for Br and ≈80 μg L−1 for Cl, corresponding to ≈0.2 mg L−1 and ≈0.4 mg L−1 of diclofenac, respectively.

The identification of unknown molecules has been one of the cornerstone applications of mass spectrometry for decades. This tutorial reviews the basics of the interpretation of electrospray ionization-based MS and MS/MS spectra in order to identify small-molecule analytes (typically below 2000 Da). Most of what is discussed in this tutorial also applies to other atmospheric pressure ionization methods like atmospheric pressure chemical/photoionization. We focus primarily on the fundamental steps of MS-based structural elucidation of individual unknown compounds, rather than describing strategies for large-scale identification in complex samples. We critically discuss topics like the detection of protonated and deprotonated ions ([M + H]+ and [M − H]−) as well as other adduct ions, the determination of the molecular formula, and provide some basic rules on the interpretation of product ion spectra. Our tutorial focuses primarily on the fundamental steps of MS-based structural elucidation of individual unknown compounds (eg, contaminants in chemical production, pharmacological alteration of drugs), rather than describing strategies for large-scale identification in complex samples. This tutorial also discusses strategies to obtain useful orthogonal information (UV/Vis, H/D exchange, chemical derivatization, etc) and offers an overview of the different informatics tools and approaches that can be used for structural elucidation of small molecules. It is primarily intended for beginning mass spectrometrists and researchers from other mass spectrometry sub-disciplines that want to get acquainted with structural elucidation are interested in some practical tips and tricks.

Lack of knowledge on the expected concentration range or insufficient linear dynamic range of the analytical method applied are common challenges for the analytical scientist. Samples that are above the upper limit of quantification are typically diluted and reanalyzed. The analysis of undiluted highly concentrated samples can cause contamination of the system, while the dilution step is time consuming and as the case for any sample preparation step, also potentially leads to precipitation, adsorption or degradation of the analytes. ispartof: Journal of Chromatography A vol:1524 pages:101-107 ispartof: location:Netherlands status: published

Sensitivity is often a critical parameter in quantitative bioanalyses in drug development. For liquid-chromatography-based methods, sensitivity can be improved by reducing the column diameter, but practical sensitivity gains are limited by the reduced sample loading capacity on small internal diameter (I.D.) columns. We developed a set-up that has overcome these limitations in sample loading capacity. The set-up uses 4 columns with gradually decreasing column diameters along the flow-path (2.1 → 1 → 0.5 → 0.15 mm). Samples are pre-concentrated on-line on a 2.1 mm I.D. trapping column and back flushed to a 1 mm I.D. UHPLC analytical column and separated. The peak(s) of interest are transferred using heartcutting to a second trapping column (0.5 mm I.D.), which is back-flushed to a 0.15 mm I.D. micro-UHPLC analytical column for orthogonal separation. The proof of concept of the set-up was demonstrated by the simultaneous analysis of midazolam and 1'-hydroxy midazolam in plasma by injection of 80 μL of protein precipitated plasma. The 4-column funnel set-up proved to be robust and resulted in a 10-50 times better sensitivity compared to a trap-elute approach and 250-500 fold better compared to direct micro-UHPLC analysis. A lower limit of quantification of 100 fg/mL in plasma was obtained for both probe compounds.

Rationale The separation of isomeric compounds with major differences in their physiochemical and pharmacokinetic properties is of particular importance in pharmaceutical R&D. However, the structural assessment and separation of these compounds with current analytical techniques and methods are still a challenge. In this study, we describe strategies to separate the various structural and stereo-isomers.Methods The separation of ten structural and stereo-isomers was investigated using Trapped and Travelling Wave ion mobility spectrometry (TIMS and TWIMS). Different strategies including adduct ion formation with Na, Li, Ag and Cs as well as fragmentation before and after the ion mobility cell were applied to separate the isomeric compounds.Results All the counter ions (in particular Na) strongly coordinated with the test analytes in all the IMS systems. The highest resolving power was achieved for the sodium and lithium adducts using TIMS-time-of-flight (TOF). However, some separation was attained on a Synapt HDMS system with its unique potential to monitor the ion mobility of the product ions. The elution order of the adduct ions was the same in all instruments, in which, unexpectedly, the para-substituted isomer of the [M + Na](+) species had the lowest collision cross section followed by the meta- and ortho-isomers.Conclusions The formation of adduct ions could facilitate the separation of structural and even stereo-isomers by generating different molecular conformations. In addition, fragmenting isomers before or after the ion mobility cell is a valuable strategy to separate and also to assess the structures of adducts and different conformers.

Aim: To evaluate alternative analytical strategies to extend the dynamic range in quantitative LC–MS/MS. Methods & results: Two approaches based on prior or no prior knowledge of expected exposure levels were evaluated. These approaches make use of two analytical strategies, which include the use of more than one injection volume or dilution of sample extract with solvents or solvent mixtures. A total of 16 compounds with varying logP values were classified into polar and nonpolar groups and used in this evaluation. From the two analytical strategies, three workflows were derived. Conclusion: All three workflows were successfully evaluated and resulted in good accuracy (80–120%) for all the compound groups.

Combining the individual analytical strengths of mass spectrometry and infrared spectroscopy, infrared ion spectroscopy is increasingly recognized as a powerful tool for small-molecule identification in a wide range of analytical applications. Mass spectrometry is itself a leading analytical technique for small-molecule identification on the merit of its outstanding sensitivity, selectivity and versatility. The foremost shortcoming of the technique, however, is its limited ability to directly probe molecular structure, especially when contrasted against spectroscopic techniques. In infrared ion spectroscopy, infrared vibrational spectra are recorded for mass-isolated ions and provide a signature that can be matched to reference spectra, either measured from standards or predicted using quantum-chemical calculations. Here we present an overview of the potential for this technique to develop into a versatile analytical method for identifying molecular structures in mass spectrometry-based analytical workflows. In this tutorial perspective, we introduce the reader to the technique of infrared ion spectroscopy and highlight a selection of recent experimental advances and applications in current analytical challenges, in particular in the field of untargeted metabolomics. We report on the coupling of infrared ion spectroscopy with liquid chromatography and present experiments that serve as proof-of-principle ex- amples of strategies to address outstanding challenges.

High-performance liquid chromatography was used in combination with infrared ion spectroscopy for the identification of positional isomers of hydroxy-atorvastatins, the primary metabolites of the drug atorvastatin. The results demonstrate the direct applicability of infrared ion spectroscopy in the field of drug metabolism and, more generally, its promising role in state-of-the-art analytical laboratories for the identification of small molecules buried in complex mixtures. In combination with chromatographic separation, infrared spectroscopy of mass-selected ions provides a promising new route for the identification of the molecular structures of unknown m/z peaks in a mass spectrum. We demonstrate that currently existing experimental protocols allow the measurement of an IR spectrum from less than 10 ng of sample obtained in a collected HPLC fraction.

Eicosanoids, including prostaglandins and thromboxanes are lipid mediators synthetized from polyunsaturated fatty acids. They play an important role in cell signaling and are often reported as inflammatory markers. LC-MS/MS is the technique of choice for the analysis of these compounds, often in combination with advanced sample preparation techniques. Here we report a head to head comparison between an electrospray ionization source (ESI) and a new atmospheric pressure ionization source (UniSpray). The performance of both interfaces was evaluated in various matrices such as human plasma, pig colon and mouse colon. The UniSpray source shows an increase in method sensitivity up to a factor 5. Equivalent to better linearity and repeatability on various matrices as well as an increase in signal intensity were observed in comparison to ESI. publisher: Elsevier articletitle: Enhanced performance for the analysis of prostaglandins and thromboxanes by liquid chromatography-tandem mass spectrometry using a new atmospheric pressure ionization source journaltitle: Journal of Chromatography A articlelink: http://dx.doi.org/10.1016/j.chroma.2016.02.055 content_type: article copyright: Copyright © 2016 Elsevier B.V. All rights reserved. ispartof: Journal of Chromatography A vol:1440 pages:260-5 ispartof: location:Netherlands status: published

The application of two-dimensional liquid chromatography (2D-LC) is gradually growing also in the area of metabolite profiling and identification. The current contribution describes a heartcut 2D-UHPLC configuration that is applied in support of drug metabolism studies in development. The setup applies four LC columns: two analytical UHPLC columns to perform the first and second dimension separations, which are both preceded by a short HPLC column operated as trapping column. The first HPLC column allows a significant online preconcentration by large volume injection. The second short HPLC column is placed between the first and second dimension columns and enables the selection of orthogonal conditions in the second dimension independent of the first dimension making the heartcutting 2D approach more generic. The value of the setup was demonstrated with selective ultraviolet chromatograms obtained for the two major hydroxylated metabolites of atorvastatin separating them from a very high biological background, originating from an injection of 4 mL feces extract, by heartcut 2D-LC. In a second application, the main metabolite of imipramine was baseline separated from some minor metabolites that were co-eluting in the first dimension, allowing accurate and sensitive quantification. A quantification limit in the attogram/mL range was achieved thanks to the injection of 200 mL diluted urine, corresponding to 100 mL urine on column.

The liver is the primary organ involved in handling of bile salts, a class of amphipathic molecules with signaling activities as well as desired and detrimental detergent actions. To allow in-depth investigation of functions of bile salts in healthy and diseased liver, the spatial distribution of bile salt species within the liver needs to be studied. Therefore, the aim of our study was to determine hepatic bile salt distribution and identify specific lipid markers that define the structural elements of the liver. Matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) was used to monitor the spatial distribution of bile salts and lipids in liver sections of rat, dog, and patients with unaffected and cholestatic parenchyma. MALDI-MSI in negative ion mode showed the local presence of a variety of bile salts, predominantly taurine-conjugates, as localized patches of varying sizes (representing the bile ducts) throughout the liver tissue. Specific molecular markers were identified for the connective tissue (phosphatidic acids, e.g., [PA (18:0_18:1)-H](-)), the liver parenchyma (phosphatidylinositols, e.g., [PI (18:0_20:4)-H](-)), and the bile ducts (hydroxylated-sulfatides, e.g., [ST-OH (18:1_24:0)-H](-)). One of these sulfatides (at m/z 906.6339) was found to be uniquely localized in a thin lining on the inside of the bile duct, colocalized with cytokeratins, and encased luminal bile salts. A similar distribution of the aforementioned sulfatide was observed, albeit in constricted ductular structures, in the liver of a patient with a mild clinical phenotype of primary sclerosing cholangitis (PSC). In contrast, sulfatides were virtually absent in the liver of patients with PSC and a severe clinical phenotype, with (atypical) cholanoids (e.g., the bile alcohol 5-cyprinolsulfate) abundant in the extra-ductular space and glyco(cheno)deoxycholic acid-3-sulfate localized to fibrotic connective tissue. The latter two molecular species were able to discriminate between healthy liver tissue (n = 3) and tissue from PSC patients with a severe clinical phenotype (n = 3). In conclusion, the distinct structural elements of the mammalian liver are characterized by specific classes of lipids. We propose that (hydroxylated-)sulfatides are specific molecular markers of the bile duct.

High-throughput simultaneous quantitative and qualitative (Quan/Qual) analysis is attractive to combine targeted with non-targeted analysis, e.g. in pharmacometabolomics and drug metabolism studies. This study aimed to investigate the possibilities and limitations of high-throughput Quan/Qual analysis by ultra-high performance liquid chromatography (UHPLC) coupled with high-resolution mass spectrometry (HRMS), to develop a widely applicable Quan/Qual UHPLC-HRMS method and to provide recommendations for Quan/Qual method development. A widely applicable 4.25-min UHPLC method for small-molecules was used to investigate and optimize mass spectrometric parameters of a Synapt G2S for Quan/Qual analysis. The method was applied on a rat metabolomics study investigating the effect of the fasting state and administration of a dosing vehicle on the rat plasma metabolic profile. scan mode with a short scan time of 30 ms. This 4.25-min Quan/Qual analysis method enabled quantification with accuracy and precision values ≤ 20% at the lowest quality control (QC) level and ≤15% at higher QC levels for 16 out of 19 tested analytes. It provided both parent m/z values and fragmentation spectra for compound identification with limited loss of chromatographic resolution and it revealed biologically relevant metabolites in its application to the metabolomics study. Quan/Qual method development requires balancing between the amount of qualitative data, the quality of the quantitative data and the analysis time. Recommendations are provided for MS resolution, scan mode, scan rate, smoothing and peak integration in Quan/Qual method development and analysis. BACKGROUND METHODS RESULTS CONCLUSION

The intestinal barrier controls intestinal permeability, and its disruption has been associated with multiple diseases. Therefore, preclinical safety biomarkers monitoring barrier integrity are essential during the development of drugs targeting the intestines, particularly if starting treatment early after onset of disease. Classical toxicology endpoints are not sensitive enough and therefore our objective was to identify non-invasive markers enabling early in vivo detection of colonic barrier perturbation. Male Sprague-Dawley rats were dosed intracolonically via the rectum, using sodium caprate or ibuprofen as tool compounds to alter barrier integrity. Several potentially translational biomarkers and probe molecules related to permeability, inflammation or tissue damage were evaluated, using various analytical platforms, including immunoassays, targeted metabolomics and highly sensitive ultra-performance liquid chromatography-tandem mass spectrometry. Several markers were identified that allow early in vivo detection of colonic barrier integrity changes, before histopathological evidence of tissue damage. The most promising permeability markers identified were plasma fluorescein isothiocyanate-dextran 4000 and a lactulose/mannitol/sucralose mixture in urine. These markers showed maximum increases over 100-fold or approximately 10-50-fold, respectively. Intracolonic administration of the above probe molecules outperformed oral administration and inflammatory or other biomarkers, such as α

Structural elucidation of metabolites is an important part during the discovery and development process of new pharmaceutical drugs. Liquid Chromatography (LC) in combination with Mass Spectrometry (MS) is usually the technique of choice for structural identification but cannot always provide precise structural identification of the studied metabolite (e.g. site of hydroxylation and site of glucuronidation). In order to identify those metabolites, different approaches are used combined with MS data including nuclear magnetic resonance, hydrogen/deuterium exchange and chemical derivatization followed by LC-MS. Those techniques are often time-consuming and/or require extra sample pre-treatment. In this paper, a fast and easy to set up tool using desorption electrospray ionization-MS for metabolite identification is presented. In the developed method, analytes in solution are simply dried on a glass plate with printed Teflon spots and then a single drop of derivatization mixture is added. Once the spot is dried, the derivatized compound is analyzed. Six classic chemical derivatizations were adjusted to work as a one drop reaction and applied on a list of compounds with relevant functional groups. Subsequently, two successive reactions on a single spot of amoxicillin were tested and the methodology described was successfully applied on an in vitro incubated alprazolam metabolite. All reactions and analyses were performed within an hour and gave useful structural information by derivatizing functional groups, making the method a time-saving and efficient tool for metabolite identification if used in addition or in some cases as an alternative to common methods.

Solid-state lipid-based formulations offer great potential for the improved oral delivery of poorly watersoluble drugs. This study investigates the use of the high surface-area clay materials, montmorillonite and laponite, as solid carriers for lipid-based formulations. The unique cationexchange properties of clay platelets were exploited to preload the ionizable hydrophobic compound, blonanserin, prior to encapsulating a drug-loaded lipid solution. Thus, solid-statelipid-based formulations with dual-loading capabilities were developed and studied. These formulations were compared with simple clay-based lipid formulations, where blonanserin was loaded in the lipid phase only. The drug release behavior of all clay-based formulations was assessed during in vitro dissolution studies under simulated gastric conditions and in vitro fast ingintestinal lipolysis studies. Montmorillonite- and laponite-based lipid formulations significantly reduced blonanserin solubilization relative to a control lipid solution and silica−lipid hybrid particles, owing to incomplete drug release from the clay cation-exchange sites. This phenomenon was replicated during in vivo pharmacokinetic studies, whereby the bioavailability of simple clay-based lipid formulations was decreased relative to controls. Importantly, the solid-state dual-loaded montmorillonite-based lipid formulation provided an optimal pharmacokinetic performance, achieving the same degree of bioavailability enhancement as the control lipid solution. These findings indicate the potential of solid-state dual-loaded clay based lipid formulations for increasing drug loading levels and enhancing the oral absorption of poorly soluble weak base compounds. Refereed/Peer-reviewed

The type and design of an ionization source can have a significant influence on the performances of a bioanalytical method. It is, therefore, of high interest to evaluate the performances of newly introduced sources to highlight their benefits and limitations in comparison to other well established sources. In this paper, liquid chromatography - mass spectrometry (LC/MS) performances of a new atmospheric pressure ionization (API) source, commercialized as UniSpray, is evaluated. The dynamic range of 24 pharmaceutical and biological compounds is compared between the new API source and electrospray ionization (ESI) for 3 different mobile phase conditions. Matrix effects are also compared with ESI on a refined selection of 19 pharmaceutical and biological compounds in 4 matrices commonly encountered in bioanalysis. A slightly better dynamic range towards lower concentrations was often observed with the new API source. Matrix effects were quite similar between the two sources with a small, but statistically significant, lower percentage of matrix effects observed for the new API source in plasma and bile in the positive ion mode, and bile in negative ion mode for ESI. Finally, the sensitivity of late eluting compounds could be improved on the new API source by post-column addition of water.

A novel quantification approach, compensating for the effect of gradient elution on the instrumental response during reversed phase high-performance liquid chromatography - inductively coupled plasma - tandem mass spectrometry analysis, has been developed and validated. As a proof of concept, diclofenac and its related compounds, including its major metabolite i.e. 4′-hydroxy-diclofenac, have been quantitatively determined in human plasma matrix. An inductively coupled plasma - tandem mass spectrometer has been applied for the interference-free determination of Cl as 35ClH2+ using H2 as a reaction gas in the collision/reaction cell. The effect of the eluent composition on the instrumental response for Cl has been thoroughly investigated for the most common organic solvents in reversed phase high-performance liquid chromatography, i.e. methanol and acetonitrile. A proper mathematical function describing the effect of the eluent composition on the sensitivity for Cl, monitored as 35ClH2+, permitted adequate correction for the otherwise detrimental effect of gradient elution for both solvents. Validation using synthetically degraded diclofenac samples spiked with its major metabolite, 4′-hydroxy-diclofenac, demonstrated appropriate accuracy (recovery for 4′-hydroxy-diclofenac between 95 and 105%) and >90% and >80% recovery for Cl using acetonitrile and methanol, respectively. When applied to spiked human plasma samples (the most important matrix in drug metabolism studies), a satisfactory accuracy (recovery of 92–98%) and precision (

The absorption, metabolism, and excretion of ibrutinib were investigated in healthy men after administration of a single oral dose of 140 mg of ¹⁴C-labeled ibrutinib. The mean (S.D.) cumulative excretion of radioactivity of the dose was 7.8% (1.4%) in urine and 80.6% (3.1%) in feces with

An online preconcentration approach was developed allowing the injection of very high volumes of biological samples, thereby greatly increasing sensitivity while maintaining LC resolution. The approach was applied to the analysis of radioactive samples from both in vitro and in vivo metabolism studies where typically the concentration of radioactivity given is often limited, while sample volume is usually not. The described online preconcentration approach reduces sample preparation and, therefore, also the risk for degradation and recovery issues often seen with offline preconcentration methods. In addition to facilitating the identification and profiling of low level metabolites within a sample, the described approach also provides robust quantitative analysis of samples derived from a range of biological matrices. The application of this approach is illustrated on real life samples from different matrices and containing drugs and metabolites with a wide variety in polarity, more specifically the analysis of extracts derived from an in vitro hepatocyte incubation, 36mL of blood/acetonitrile (1/1, v/v; 28dpm/mL) and 72mL of urine/methanol (9/1, v/v; 208dpm/mL).

Janssen Research & Development, a Division of Janssen Pharmaceutica N.V., Belgium The role of MS in peptide quantification For quantitative analysis of small-molecule drugs, LC–MS is considered the gold standard. However, focusing on peptide drugs, the majority of peptide development programs still rely on immunoassays for evaluation of exposure of the peptide drug in study samples. The advantages of these assays are the unrivaled sensitivity and the high throughput. Selectivity issues and method development times are, on the other hand, often rate-limiting. LC–MS technology can offer an alternative to improve selectivity and reduce method development time (no reagent preparation needed), but at the expense of lower sensitivity and throughput. Therefore, in our portfolio, for early peptide drug programs, quantitative workflows with LC–MS are preferred to address the short cycle times requested at this stage. In development and also with the aim to evaluate anti-drug antibody response, immunological assays are primarily preferred. An LC–MS assay can then be requested as an orthogonal assay to confirm the selectivity of the immunoassay or to address specific issues. In rare cases, the immunological assay is confronted with considerable challenges and LC–MS(/MS) can be developed faster and with more confidence to support the program.

Quantitative determination of the candidate drug molecule and its metabolites in biofluids and tissues is an inevitable step in the development of new pharmaceuticals. Because of the time-consuming and expensive nature of the current standard technique for quantitative metabolite profiling, i.e., radiolabeling followed by high-performance liquid chromatography (HPLC) with radiodetection, the development of alternative methodologies is of great interest. In this work, a simple, fast, sensitive, and accurate method for the quantitative metabolite profiling of an amino group containing drug (levothyroxine) and its metabolites in human plasma, based on precolumn derivatization followed by HPLC-inductively coupled plasma mass spectrometry (ICPMS), was developed and validated. To introduce a suitable “heteroelement” (defined here as an element that is detectable with ICPMS), an inexpensive and commercially available reagent, tetrabromophthalic anhydride (TBPA) was used for the derivatization of free NH2-groups....

Aims Canagliflozin is a recently approved drug for use in the treatment of type 2 diabetes. The potential for canagliflozin to cause clinical drug-drug interactions (DDIs) was assessed. Methods DDI potential of canagliflozin was investigated using in vitro test systems containing drug metabolizing enzymes or transporters. Basic predictive approaches were applied to determine potential interactions in vivo. A physiologically-based pharmacokinetic (PBPK) model was developed and clinical DDI simulations were performed to determine the likelihood of cytochrome P450 (CYP) inhibition by canagliflozin. Results Canagliflozin was primarily metabolized by uridine 5'-diphospho-glucuronosyltransferase 1A9 and 2B4 enzymes. Canagliflozin was a substrate of efflux transporters (P-glycoprotein, breast cancer resistance protein and multidrug resistance-associated protein-2) but was not a substrate of uptake transporters (organic anion transporter polypeptide isoforms OATP1B1, OATP1B3, organic anion transporters OAT1 and OAT3, and organic cationic transporters OCT1, and OCT2). In inhibition assays, canagliflozin was shown to be a weak in vitro inhibitor (IC50 ) of CYP3A4 (27 μmol l -1 , standard error [SE] 4.9), CYP2C9 (80 μmol l -1 , SE 8.1), CYP2B6 (16 μmol l-1 , SE 2.1), CYP2C8 (75 μmol l -1 , SE 6.4), P-glycoprotein (19.3 μmol l -1 , SE 7.2), and multidrug resistance-associated protein-2 (21.5 μmol l -1 , SE 3.1). Basic models recommended in DDI guidelines (US Food & Drug Administration and European Medicines Agency) predicted moderate to low likelihood of interaction for these CYPs and efflux transporters. PBPK DDI simulations of canagliflozin with CYP probe substrates (simvastatin, S-warfarin, bupropion, repaglinide) did not show relevant interaction in humans since mean areas under the concentration-time curve and maximum plasma concentration ratios for probe substrates with and without canagliflozin and its 95% CIs were within 0.80-1.25. Conclusions In vitro DDI followed by a predictive or PBPK approach was applied to determine DDI potential of canagliflozin. Overall, canagliflozin is neither a perpetrator nor a victim of clinically important interactions.

A new atmospheric pressure ionization (API) source, viz. UniSpray, was evaluated for mass spectrometry (MS) analysis of pharmaceutical compounds by head-to-head comparison with electrospray ionization (ESI) on the same high-resolution MS system. The atmospheric pressure ionization source is composed of a grounded nebulizer spraying onto a high voltage, cylindrical stainless steel target. Molecules are ionized in a similar fashion to electrospray ionization, predominantly producing protonated or deprotonated species. Adduct formation (e.g., proton and sodium adducts) and in-source fragmentation is shown to be almost identical between the two sources. The performance of the new API source was compared with electrospray by infusion of a mix of 22 pharmaceutical compounds with a wide variety of functional groups and physico-chemical properties (molecular weight, logP, and pKa) in more than 100 different conditions (mobile phase strength, solvents, pH, and flow rate). The new API source shows an intensity gain of a factor 2.2 compared with ESI considering all conditions on all compounds tested. Finally, some hypotheses on the ionization mechanism, similarities, and differences with ESI, are discussed. Graphical Abstract ᅟ.

Background: Peptides represent a growing class of potential drugs. Information on metabolic clearance can be valuable for peptide drug development but different challenges are encountered with the identification of peptide metabolites in comparison to the process used for small-molecule therapeutics. Results: Enfuvirtide was selected as a test compound and dosed intravenously at 2 mg/kg to rats. Plasma samples were collected and analyzed on two different quadrupole-TOF instruments in positive and negative ion modes. Different post-acquisition processing tools were evaluated to identify the metabolites of a peptide drug in the presence of an in vivo matrix. Charge state filtering and ion mobility extraction were applied to reduce the matrix background and combined with more comprehensive software tools generally used for large molecule analyses as well as tools designed for small-molecule metabolite identification work. Conclusion: Both ion mobility spectrometry and charge state filtration proved to be successful in extracting peptide ions and significantly reducing background signals. Both small- and large-molecule software tools contain specific capabilities that could be usefully combined in a single package for peptide metabolite identification.

Background: There is considerable interest in the determination of relative abundances of human metabolites in plasma (and potentially excreta) with reasonable accuracy early on in the drug development process in order to make scientifically sound decisions with regard to the presence of potentially active or toxic disproportionate metabolites. At this point, authentic metabolite standards are generally not available. Results: A new methodology is proposed for the estimation of metabolite concentrations in the absence of authentic standards. A reference sample containing radiolabeled metabolites of interest is produced by incubating the 14C-labeled drug in vitro, and mixed with a sample to be quantitated containing the unlabeled metabolites. The 12C/14C isotope ratio is measured with high-resolution ESI–MS for each metabolite, and used as a basis for quantitation of the cold metabolite based on the concentration of radioactive metabolite, determined from independent analysis of the radioactive sample with LC-radiochemical detection. The 14C-labeled metabolite serves as an isotopically labeled internal standard, which corrects for any variations in injection volume, sample preparation, MS intensity drift, matrix effects and/or saturation of electrospray ionization. The approach was validated by the analysis of solutions containing variable amounts of the analyte with a fixed amount of radioactive standard on a QToF Synapt® G2 MS system. The same methodology was also successfully applied to first-in-human plasma samples analyzed on a LTQ-Orbitrap®. Conclusion: The metabolite abundances obtained by 12C/14C isotope ratio measurements showed suitable accuracy and precision and were very close to those obtained with matrix mixing. The parent drug concentrations also corresponded well with the bioanalytical results obtained with a validated LC–MS/MS method.

Travelling wave ion mobility spectrometry - mass spectrometry (TWIMS-MS) was evaluated as a tool for structural identification of metabolites of small molecule drugs in cases where the exact position of the biotransformation could not be identified by conventional tandem mass spectrometry. Test sets of compounds containing biotransformations at aromatic positions were analyzed. These present a problem for traditional MS methods since an atomic level localization of the biotransformation cannot normally be determined from MS(n) spectra. In addition to ion mobility measurements of the intact metabolite ions, ion mobility measurements of product ions were also made and the results compared with calculated values. This approach reduces the complexity of the problem, making theoretical calculations easier and more predictable when a modeled collision cross section (CCS) is required. A good relative correspondence between theoretical and measured CCSs was obtained allowing the identification of the exact position of the biotransformation. It was also demonstrated that authentic standards with substructures identical to those in the unknown can be used to assign the exact position of the biotransformation. In this approach the identification was based on the comparison of the drift times or CCSs for product ions of the standard, with those of the same product ions in the unknown.

The aim of this work was speciation analysis of metabolites in feces samples collected within a clinical study during which a bromine-containing anti-tuberculosis drug (TMC207) was administered to patients with multi-drug resistant tuberculosis infection. Owing to slow elimination of the drug, no (14)C label was used within this study. Quantification of the bromine species was accomplished using high performance liquid chromatography coupled to inductively coupled plasma-mass spectrometry (HPLC/ICP-MS) in combination with on-line isotope dilution (on-line ID), while structural elucidation of the species was performed using HPLC coupled to electrospray ionization-mass spectrometry. The ICP-MS-based method developed shows a good intra- and inter-day reproducibility (relative standard deviation = 3.5%, N = 9); the limit of detection (1.5 mg TMC207 L(-1)) is of the same order of magnitude as that for HPLC/radiodetection; the dynamic range of the method covers more than two orders of magnitude. Furthermore, the column recovery was demonstrated to be quantitative (recoveries between 90.6% and 99.5%). Based on the excellent figures of merit, the "cold" HPLC/ICP-MS approach could be deployed for the actual human in vivo metabolism study, such that exposure of the human volunteers to the (14)C radiolabel was avoided.

Bioassay-guided isolation was performed on the leaves of Apodytes dimidiata E. Mey. Ex Arn. (Icacinaceae), based on previously demonstrated activity against Leishmania. Six saponins never isolated from nature before were elucidated with LCMS/MS, GCMS and 1D and 2D NMR. The compounds apodytine AF are responsible at least in part for the antiprotozoal activity, but also possess haemolytic activity and display antiangiogenic activity in the rat aorta ring assay, an effect which may be due to a non-selective toxicity.