Search

Benzimidazoles anthelmintics, which enter into environment primarily through excretion in the feces or urine of treated animals, can affect various organisms and disrupt ecosystem balance. The present study was designed to test the phytotoxicity and biotransformation of the three benzimidazole anthelmintics albendazole (ABZ), fenbendazole (FBZ) and flubendazole (FLU) in the harebell (Campanula rotundifolia). This meadow plant commonly grows in pastures and comes into contact with anthelmintics through the excrements of treated animals. Suspensions of harebell cells in culture medium were used as an in vitro model system. ABZ, FLU and FBZ were not found to be toxic for harebell cells, which were able to metabolize ABZ, FLU and FBZ via the formation of a wide scale of metabolites. Ultrahigh-performance liquid chromatography coupled with high mass accuracy tandem mass spectrometry (UHPLC-MS/MS) led to the identification of 24, 18 and 29 metabolites of ABZ, FLU and FBZ, respectively. Several novel metabolites were identified for the first time. Based on the obtained results, the schemes of the metabolic pathways of these anthelmintics were proposed. Most of these metabolites can be considered deactivation products, but a substantial portion of them may readily be decomposed to biologically active substances which could negatively affect ecosystems., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Reversed-phase ultrahigh-performance liquid chromatography (RP-UHPLC) method using two 15cm sub-2μm particles octadecylsilica gel columns is developed with the goal to separate and unambiguously identify a large number of lipid species in biological samples. The identification is performed by the coupling with high-resolution tandem mass spectrometry (MS/MS) using quadrupole - time-of-flight (QTOF) instrument. Electrospray ionization (ESI) full scan and tandem mass spectra are measured in both polarity modes with the mass accuracy better than 5ppm, which provides a high confidence of lipid identification. Over 400 lipid species covering 14 polar and nonpolar lipid classes from 5 lipid categories are identified in total lipid extracts of human plasma, human urine and porcine brain. The general dependences of relative retention times on relative carbon number or relative double bond number are constructed and fit with the second degree polynomial regression. The regular retention patterns in homologous lipid series provide additional identification point for UHPLC/MS lipidomic analysis, which increases the confidence of lipid identification. The reprocessing of previously published data by our and other groups measured in the RP mode and ultrahigh-performance supercritical fluid chromatography on the silica column shows more generic applicability of the polynomial regression for the description of retention behavior and the prediction of retention times. The novelty of this work is the characterization of general trends in the retention behavior of lipids within logical series with constant fatty acyl length or double bond number, which may be used as an additional criterion to increase the confidence of lipid identification., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The present in vitro study was designed to test and compare anthelmintic activity, hepatotoxicity, and biotransformation of four selected aminoacetonitrile derivatives (AADs): monepantel (MOP, anthelmintic approved for the treatment), AAD-970, AAD-1154, and AAD-1336. Micro-agar larval development test, MTT test of cytotoxicity, and biotransformation study coupled with Ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) technique were used for this purpose. Larvae of two Haemonchus contortus strains (drug susceptible and multi-drug resistant) and primary cultures of rat and ovine hepatocytes served as model systems. All AADs (including MOP) exhibited significant larvicidal effect in H. contortus susceptible as well as multi-resistant strains, much higher than those of reference anthelmintics thiabendazole and flubendazole. AAD-1154 provides the best results for most tested parameters among all AADs in this study. The cytotoxicity test showed that all AADs can be considered as nontoxic for hepatocytes. In the biotransformation study, Phase I and Phase II metabolites of AADs were identified and schemes of possible metabolic pathways in ovine hepatocytes were proposed. Biotransformation of MOP was much more extensive than biotransformation of other AADs. Based on obtained results, AAD-1154 and AAD-1336 can be considered as promising candidates for further in vivo testing., (Copyright © 2015 John Wiley & Sons, Ltd.)

Rationale: Triacylglycerols (TG) contain three fatty acyls attached to the glycerol backbone in stereochemically numbered positions sn-1, 2 and 3. Isobaric TG with exchanged fatty acyl chains in positions sn-1/3 vs. sn-2 are referred to as regioisomers and the determination of their regioisomeric ratios is important for nutrition purposes., Methods: Differential mobility spectrometry (DMS) coupled to electrospray ionization mass spectrometry (ESI-MS) is applied for the separation of simple unsaturated TG regioisomers extracted from porcine adipose tissue using their silver-ion molecular adducts., Results: Four pairs of TG regioisomers containing combinations of unsaturated and saturated fatty acyl chains are successfully separated using DMS with 1-butanol or 1-propanol as the chemical modifier. Various experimental parameters are carefully optimized, such as the separation and compensation voltages applied to DMS electrodes, the type and flow rate of chemical modifier and the dwell time of analyte ions in the DMS cell. The optimized DMS approach is used for the characterization of TG regioisomers in less than one minute, compared to tens of minutes typical for silver-ion or reversed-phase high-performance liquid chromatography/mass spectrometry approaches., Conclusions: The application of this method for the characterization of TG regioisomers in porcine adipose tissue shows the method suitability for analyses of other animal fats.

Rationale: Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is used for the fast qualitative and quantitative analysis of phosphatidylcholines (PC). Fatty acyl chain lengths and the number of double bonds (DB) affect relative responses of PC; hence the determination of correction factors of individual PC is important for the accurate quantitation. The signal intensity in MALDI-MS strongly depends on the matrix; therefore, the following matrices typically used in lipidomics are studied in the present work: 2,5-dihydroxybenzoic acid (DHB), 1,5-diaminonaphthalene (DAN) and 9-aminoacridine (9AA)., Methods: Series of PC with various fatty acyl chain lengths are synthesized for this study. PC concentrations over two orders of magnitude are studied with MALDI-MS. These experiments provide sets of calibration curves for each of the synthesized PC and the further analysis of parameters of calibration curves is performed., Results: Correction factors for PC decrease with increasing fatty acyl chain length for all matrices. These dependences are steeper for unsaturated PC than for saturated ones. MALDI matrices also have a significant effect on this dependence. The weakest dependence on fatty acyl chain length is found for saturated PC in 9AA. In the case of the other matrices, the effect of fatty acyl chain length on the response is essential for both saturated and unsaturated PC. Calibration curves and parameters of calibration curves for both saturated and monounsaturated PC are fitted by a linear function with regression coefficients decreasing in the order 9AA > DAN > DHB., Conclusions: Differences in relative responses for PC in MALDI-MS measurements must be taken into account for accurate quantitation. Parameters of calibration curves can be used for the determination of PC concentrations using a single internal standard (IS). This method gives good results for the 9AA matrix, but the reproducibility of measurements for the DHB and DAN matrices is lower and the method can be used for a rough estimation only. These matrices are less convenient for the quantitation of PC., (Copyright © 2015 John Wiley & Sons, Ltd.)

The characterization of differences among polar lipid classes in tumors and surrounding normal tissues of 20 kidney cancer patients is performed by hydrophilic interaction liquid chromatography (HILIC) coupled to electrospray ionization mass spectrometry (ESI-MS). The detailed analysis of identified lipid classes using relative abundances of characteristic ions in negative- and positive-ion modes is used for the determination of more than 120 individual lipid species containing attached fatty acyls of different chain length and double bond number. Lipid species are described using relative abundances, providing a better visualization of lipidomic differences between tumor and normal tissues. The multivariate data analysis methods using unsupervised principal component analysis (PCA) and supervised orthogonal partial least square (OPLS) are used for the characterization of statistically significant differences in identified lipid species. Ten most significant up- and down-regulated lipids in OPLS score plots are also displayed by box plots. A notable increase of relative abundances of lipids containing four and more double bonds is detected in tumor compared to normal tissues., (Copyright © 2015 Elsevier B.V. All rights reserved.)

New analytical approach for high-throughput and comprehensive lipidomic analysis of biological samples using ultrahigh-performance supercritical fluid chromatography (UHPSFC) with electrospray ionization-mass spectrometry (ESI-MS) is presented in this work as an alternative approach to established shotgun MS or high-performance liquid chromatography-MS. The lipid class separation is performed by UHPSFC method based on 1.7 μm particle-bridged ethylene hybrid silica column with a gradient of methanol-water-ammonium acetate mixture as a modifier. All parameters of UHPSFC conditions are carefully optimized and their influence on the chromatographic behavior of lipids is discussed. The final UHPSFC/ESI-MS method enables a fast separation of 30 nonpolar and polar lipid classes within 6 min analysis covering 6 main lipid categories including fatty acyls, glycerolipids, glycerophospholipids, sphingolipids, sterols, and prenols. Individual lipid species within lipid classes are identified based on positive and negative-ion full-scan and tandem mass spectra measured with high mass accuracy and high resolving power. Developed UHPSFC/ESI-MS method is applied for the analysis of porcine brain extract as a complex lipidomic sample, where 24 lipid classes containing 436 lipid species are identified. The method is validated for the quantitative analysis of lipid species in biological tissues using internal standards for each lipid class. This high-throughput, comprehensive and accurate UHPSFC/ESI-MS method is suitable for the lipidomic analysis of large sample sets in the clinical research.

A new continuous comprehensive two-dimensional liquid chromatography-electrospray ionization mass spectrometry method has been developed for the lipidomic characterization of complex biological samples. The reversed-phase ultra-high-performance liquid chromatography with a C18 column (150 mm × 1 mm, 1.7 μm) used in the first dimension makes the separation of numerous lipid species differing in their hydrophobic part of the molecule, mainly fatty acyl chain lengths and the number and positions of double bonds, possible. Coeluted lipid species in the first dimension are resolved by the fast hydrophilic interaction liquid chromatography separation (50 mm × 3 mm, 2.7 μm, core-shell particles) of lipid classes according to their different polarities in the second dimension. Retention times in both dimensions, accurate m/z values, and tandem mass spectra provide high confidence in the identification of lipid species. The retention behavior of individual lipids in reversed-phase mode follows the equivalent carbon number pattern, which provides an additional tool for unambiguous identification. This analytical method is applied for the lipidomic characterization of total lipid extracts of human plasma and porcine brain samples, which resulted in the identification of 143 lipid species from four lipid categories and ten lipid classes.

Differences among lipidomic profiles of healthy volunteers, obese people and three groups of cardiovascular disease (CVD) patients are investigated with the goal to differentiate individual groups based on the multivariate data analysis (MDA) of lipidomic data from plasma, erythrocytes and lipoprotein fractions of more than 50 subjects. Hydrophilic interaction liquid chromatography on ultrahigh-performance liquid chromatography (HILIC-UHPLC) column coupled with electrospray ionization mass spectrometry (ESI-MS) is used for the quantitation of four classes of polar lipids (phosphatidylethanolamines, phosphatidylcholines, sphingomyelins and lysophosphatidylcholines), normal-phase UHPLC-atmospheric pressure chemical ionization MS (NP-UHPLC/APCI-MS) is applied for the quantitation of five classes of nonpolar lipids (cholesteryl esters, triacylglycerols, sterols, 1,3-diacylglycerols and 1,2-diacylglycerols) and the potential of matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is tested for the fast screening of all lipids without a chromatographic separation. Obtained results are processed by unsupervised (principal component analysis) and supervised (orthogonal partial least squares) MDA approaches to highlight the largest differences among individual groups and to identify lipid molecules with the highest impact on the group differentiation., (Copyright © 2015 Elsevier B.V. All rights reserved.)

A novel normal-phase (NP) ultrahigh-performance liquid chromatography-mass spectrometry (UHPLC/MS) method is developed for a separation and quantitation of nonpolar lipid classes occurring in human plasma, erythrocytes and plasma lipoprotein fractions. The baseline class separation of cholesteryl esters (CE), cholesterol, triacylglycerols (TG), regioisomers of 1,2- and 1,3-diacylglycerols (DG) and 1-monoacylglycerols (1-MG) is achieved using an optimized hexane - 2-propanol-acetonitrile mobile phase within 18min for all nonpolar lipid classes or only 9min excluding monoacylglycerols not detected in studied samples. The determination of individual nonpolar lipid classes is performed by the response factor approach and the use of dioleoyl ethylene glycol as a single internal standard. Polar lipid classes, such as phosphatidylglycerols (PG), phosphatidylethanolamines (PE), phosphatidylcholines (PC), sphingomyelins (SM) and lysophosphatidylcholines (LPC), are separated by hydrophilic interaction liquid chromatography (HILIC) using 5mmol/L aqueous ammonium acetate-methanol-acetonitrile gradient within 13minutes. The quantitation of polar lipid classes is done by a similar approach as for nonpolar lipid classes, but a different internal standard (sphingosyl PE d17:1/12:0) is used. The complementary information on fatty acyl profiles after the transesterification of the total lipid extract is obtained by gas chromatography with flame ionization detection (GC/FID). The applicability of developed methodology for fast and comprehensive characterization of blood lipidome is illustrated on samples of human plasma, erythrocytes, high-density lipoprotein (HDL), low-density lipoprotein (LDL) and very low-density lipoprotein (VLDL) fractions., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The comprehensive approach for the lipidomic characterization of human breast cancer and surrounding normal tissues is based on hydrophilic interaction liquid chromatography (HILIC)-electrospray ionization mass spectrometry (ESI-MS) quantitation of polar lipid classes of total lipid extracts followed by multivariate data analysis using unsupervised principal component analysis (PCA) and supervised orthogonal partial least square (OPLS). This analytical methodology is applied for the detailed lipidomic characterization of ten patients with the goal to find the statistically relevant differences between tumor and normal tissues. This strategy is selected for better visualization of differences, because the breast cancer tissue is compared with the surrounding healthy tissue of the same patient, therefore changes in the lipidome are caused predominantly by the tumor growth. A large increase of total concentrations for several lipid classes is observed, including phosphatidylinositols, phosphatidylethanolamines, phosphatidylcholines, and lysophosphatidylcholines. Concentrations of individual lipid species inside the abovementioned classes are also changed, and in some cases, these differences are statistically significant. PCA and OPLS analyses enable a clear differentiation of tumor and normal tissues based on changes of their lipidome. A notable decrease of relative abundances of ether and vinylether (plasmalogen) lipid species is detected for phosphatidylethanolamines, but no difference is apparent for phosphatidylcholines.

Monepantel (MOP) is a new anthelmintic drug intended for the treatment and control of gastrointestinal roundworms (nematodes) infection and associated disease in sheep. The aim of our study was to find out metabolic pathways of MOP in sheep in vivo and in its parasite Haemonchus contortus ex vivo. MOP biotransformation in two H. contortus strains with different sensitivity to anthelmintics was also compared. Ultra high-performance liquid chromatography with tandem mass spectrometry (UHPLC-MS/MS) technique is used for the identification of MOP metabolites in ovine urine, faeces, and nematodes. MOP biotransformation study in sheep in vivo led to the identification of 13 MOP metabolites; 7 of them have not been described previously in in vitro study. The study of MOP biotransformation in H. contorus ex vivo reveals four MOP metabolites. The nitrile hydrolysis as a new biotransformation pathway in helminths ex vivo is reported here for the first time. Unlike sheep, H. contorus nematodes are not able to metabolize MOP via phase II biotransformation. Nematodes of resistant White river (WR) strain form more types of MOP metabolites than nematodes of sensitive inbred susceptible Edinburgh (ISE) strain. Based on obtained results, schemes of metabolic pathways of MOP in sheep and nematodes are proposed., (Copyright © 2014 John Wiley & Sons, Ltd.)

The combination of ultrahigh-resolution mass spectrometry imaging (UHRMSI) and ultrahigh-performance liquid chromatography coupled with tandem mass spectrometry (UHPLC/MS/MS) was used for the identification and the spatial localization of atorvastatin (AT) and its metabolites in rat tissues. Ultrahigh-resolution and high mass accuracy measurements on a matrix-assisted laser desorption/ionization (MALDI)-Orbitrap mass spectrometer allowed better detection of desired analytes in the background of matrix and endogenous compounds. Tandem mass spectra were also used to confirm the identification of detected metabolites in complex matrices. The optimization of sample preparation before imaging experiments included the tissue cryogenic sectioning (thickness 20 μm), the transfer to stainless steel or glass slide, and the selection of suitable matrix and its homogenous deposition on the tissue slice. Thirteen matrices typically used for small molecule analysis, e.g., 2,5-dihydroxybenzoic acid (DHB), 1,5-diaminonaphthalene (DAN), 9-aminoacridine (AA), etc., were investigated for the studied drug and its metabolite detection efficiency in both polarity modes. Particular matrices were scored based on the strength of extracted ion current (EIC), relative ratio of AT molecular adducts, and fragment ions. The matrix deposition on the tissue for the most suitable matrices was done by sublimation to obtain the small crystal size and to avoid local variations in the ionization efficiency. UHPLC/MS profiling of drug metabolites in adjacent tissue slices with the previously optimized extraction was performed in parallel to mass spectrometry imaging (MSI) measurements to obtain more detailed information on metabolites in addition to the spatial information from MSI. The quantitation of atorvastatin in rat liver, serum, and feces was also performed.

High-performance liquid chromatography (HPLC) coupled with electrospray ionization mass spectrometry (ESI-MS) was applied for the analysis of nabumetone metabolites during the biotransformation in minipigs. In addition to known phase I metabolites, the identification of phase II metabolites was achieved on the basis of their full-scan mass spectra and subsequent MS(n) analysis using both positive-ion and negative-ion ESI mode. Some phase I metabolites are conjugated with both glucuronide acid and glycine, which is quite unusual type of phase II metabolite not presented so far for nabumetone. These metabolites were found in small intestine content, but they were absent in minipigs urine., (Copyright © 2013 Elsevier B.V. All rights reserved.)

A systematic study of the retention behavior of isomeric triacylglycerols (TGs) in silver-ion HPLC on a ChromSpher Lipids column has been performed between 10 to 40°C using the most widespread hexane- and dichloromethane-based mobile phases. The randomization of mono-acyl TG standards and the random esterification of glycerol with fatty acids are employed to produce mixtures of TG isomers. The mobile phase composition has no influence on the general retention pattern, but significant differences in the retention order of double bond (DB) positional isomers in hexane and dichloromethane mobile phases are described and compared with the previous literature data. Saturated TGs with fatty acyl chain length from C7:0 to C22:0 are partially separated using the hexane mobile phase but not at all with the dichloromethane mobile phase. The hexane mobile phase enables at least partial resolution of TG regioisomers with up to seven DBs, while the resolution of only ALA/AAL and ALnA/AALn isomers is achieved with the dichloromethane mobile phase. The effect of temperature differs significantly depending on the mobile phase composition. Retention times of TGs increase with increasing temperature in the hexane mobile phase, while an opposite effect is observed for the dichloromethane mobile phase., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Ultrahigh-performance liquid chromatography coupled with high-mass-accuracy tandem mass spectrometry (UHPLC-MS-MS) has been used for elucidation of the structures of oxidation products of atorvastatin (AT), one of the most popular commercially available drugs. The purpose of the study was identification of AT metabolites in rat hepatocytes and comparison with electrochemically generated oxidation products. AT was incubated with rat hepatocytes for 24 h. Electrochemical oxidation of AT was performed by use of a three-electrode off-line system with a glassy carbon working electrode. Three supporting electrolytes (0.1 mol L(-1) H2SO4, 0.1 mol L(-1) HCl, and 0.1 mol L(-1) NaCl) were tested, and dependence on pH was also investigated. AT undergoes oxidation by a single irreversible process at approximately +1.0 V vs. Ag/AgCl electrode. The results obtained revealed a simple and relatively fast way of determining the type of oxidation and its position, on the basis of characteristic neutral losses (NLs) and fragment ions. Unfortunately, different products were obtained by electrochemical oxidation and biotransformation of AT. High-mass-accuracy measurement combined with different UHPLC-MS-MS scans, for example reconstructed ion-current chromatograms, constant neutral loss chromatograms, or exact mass filtering, enable rapid identification of drug-related compounds. β-Oxidation, aromatic hydroxylation of the phenylaminocarbonyl group, sulfation, AT lactone and glycol formation were observed in rat biotransformation samples. In contrast, a variety of oxidation reactions on the conjugated skeleton of isopropyl substituent of AT were identified as products of electrolysis.

Lipids form a significant part of animal organs and they are responsible for important biological functions, such as semi-permeability and fluidity of membranes, signaling activity, anti-inflammatory processes, etc. We have performed a comprehensive nontargeted lipidomic characterization of porcine brain, heart, kidney, liver, lung, spinal cord, spleen, and stomach using hydrophilic interaction liquid chromatography (HILIC) coupled to electrospray ionization mass spectrometry (ESI/MS) to describe the representation of individual lipid classes in these organs. Detailed information on identified lipid species inside classes are obtained based on relative abundances of deprotonated molecules [M-H](-) in the negative-ion ESI mass spectra, which provides important knowledge on phosphatidylethanolamines and their different forms of fatty acyl linkage (ethers and plasmalogens), phosphatidylinositols, and hexosylceramides containing nonhydroxy- and hydroxy-fatty acyls. The detailed analysis of identified lipid classes using reversed-phase liquid chromatography in the second dimension was performed for porcine brain to determine more than 160 individual lipid species containing attached fatty acyls of different acyl chain length, double-bond number, and positions on the glycerol skeleton. The fatty acid composition of porcine organs is determined by gas chromatography with flame ionization detection after the transesterification with sodium methoxide.

Nabumetone is a non-acidic, nonsteroidal anti-inflammatory prodrug. Following oral administration, the prodrug is converted in the liver to 6-methoxy-2-naphthylacetic acid (6-MNA), which was found to be the principal metabolite responsible for the NSAID effect. The pathway of nabumetone transformation to 6-MNA has not been clarified, with no intermediates between nabumetone and 6-MNA having been identified to date. In this study, a new, as yet unreported phase I metabolite was discovered within the evaluation of nabumetone metabolism by human and rat liver microsomal fractions. Extracts from the biomatrices were subjected to chiral LLE-HPLC-PDA and achiral LLE-UHPLC-MS/MS analyses to elucidate the chemical structure of this metabolite. UHPLC-MS/MS experiments detected the presence of a structure corresponding to elemental composition C15H16O3, which was tentatively assigned as a hydroxylated nabumetone. Identical nabumetone and HO-nabumetone UV spectra obtained from the PDA detector ruled out the presence of the hydroxy group in the aromatic moiety of nabumetone. Hence, the most likely structure of the new metabolite was 4-(6-methoxy-2-naphthyl)-3-hydroxybutan-2-one (3-hydroxy nabumetone). To confirm this structure, the standard of this nabumetone metabolite was synthesized, its spectral (UV, CD, NMR, MS/MS) and retention properties on chiral and achiral chromatographic columns were evaluated and compared with those of the authentic nabumetone metabolite. To elucidate the subsequent biotransformation of 3-hydroxy nabumetone, the compound was used as a substrate in incubation with human and rat liver microsomal fraction. A number of 3-hydroxy nabumetone metabolites (products of conjugation with glucuronic acid, O-desmethylation, carbonyl reduction and their combination) were discovered in the extracts from the incubated microsomes using LLE-HPLC-PDA-MS/MS experiments. On the other hand, when 3-hydroxy nabumetone was incubated with isolated rat hepatocytes, 6-MNA was detected as the principal metabolite of 3-hydroxy nabumetone. Hence, 3-hydroxy nabumetone could be the missing link in nabumetone biotransformation to 6-MNA (i.e. nabumetone→3-hydroxy nabumetone→6-MNA)., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Ivermectin (IVE), one of the most important anthelmintics, is often used in the treatment of haemonchosis in ruminants. The objective of our work was (1) to find and identify phase I and II metabolites of IVE formed by the Barber's pole worm (Haemonchus contortus), and (2) to compare IVE metabolites in helminths with IVE biotransformation in sheep (Ovis aries) as host species. Ultrahigh-performance liquid chromatography/tandem mass spectrometry (UHPLC/MS/MS) was used for this purpose. During in vitro incubations, microsomes (from adult worms or from ovine liver) and a primary culture of ovine hepatocytes were incubated with IVE. In the ex vivo study, living H. contortus adults were incubated in the presence of 1 μM IVE for 24 h. The results showed that the H. contortus enzymatic system is not able to metabolize IVE. On the other hand, 7 different phase I as well as 9 phase II IVE metabolites were detected in ovine samples using UHPLC/MS/MS analyses. Most of these metabolites have not been described before. Haemonchus contortus is not able to deactivate IVE through biotransformation; therefore, biotransformation does not contribute to the development of IVE-resistance in the Barber's pole worm.

In this work, the first systematic characterization of triacylglycerol (TG) enantiomers in real samples using chiral high-performance liquid chromatography (HPLC) with atmospheric pressure chemical ionization mass spectrometry (APCI-MS) is performed. Our chiral HPLC/APCI-MS method is based on the use of two cellulose-tris-(3,5-dimethylphenylcarbamate) columns connected in series using a gradient of hexane-2-propanol mobile phase. All TG enantiomers containing 1-8 DBs and different fatty acyl chain lengths are separated using our chiral HPLC method except for TGs having a combination of saturated and di- or triunsaturated fatty acyls in sn-1 and sn-3 positions. In our work, the randomization reaction of monoacyl TG standards is used for the preparation of all TG enantiomers and regioisomers in a mixture, while the stereospecific esterification of 1,2- or 2,3-isopropylidene-sn-glycerols by selected fatty acids is used for the synthesis of TG enantiomers. The composition of TG enantiomers and regioisomers in hazelnut oil and human plasma samples is determined. Unsaturated fatty acids are preferentially esterified in sn-2 position in hazelnut oil, while no significant preference of saturated or unsaturated fatty acyls is observed in case of human plasma sample. Fatty acids with the higher number of DBs are preferred in sn-1 position of TG enantiomers in hazelnut oil unlike to moderate sn-3 preference in human plasma. The characterization of cholesteryl esters from TG fraction of human plasma sample using our chiral HPLC/APCI-MS method is presented as well.

Monepantel (MOP) belongs to a new class of anthelmintic drugs known as aminoacetonitrile derivatives. It was approved for use in veterinary practice in Czech Republic in 2011. So far, biotransformation and transport of MOP in target animals have been studied insufficiently, although the study of metabolic pathways of anthelmintics is very important for the efficacy of safety of therapy and evaluation of the risk of drug-drug interactions. The aim of this study was to identify MOP metabolites and to suggest the metabolic pathways of MOP in sheep. For this purpose, primary culture of ovine hepatocytes was used as a model in vitro system. After incubation, medium samples and homogenates of hepatocytes were extracted separately using solid-phase extraction. Analysis was performed using a hybrid quadrupole-time-of-flight analyzer with respect to high mass accuracy measurements in full scan and tandem mass spectra for the confirmation of an elemental composition. The obtained results revealed S-oxidation to sulfoxide and sulfone and arene hydroxylation as MOP phase I biotransformations. From phase II metabolites, MOP glucuronides, sulfates, and acetylcysteine conjugates were found. Based on the obtained results, a scheme of the metabolic pathway of MOP in sheep has been proposed.

The identification and quantitation of a wide range of lipids in complex biological samples is an essential requirement for the lipidomic studies. High-performance liquid chromatography-mass spectrometry (HPLC/MS) has the highest potential to obtain detailed information on the whole lipidome, but the reliable quantitation of multiple lipid classes is still a challenging task. In this work, we describe a new method for the nontargeted quantitation of polar lipid classes separated by hydrophilic interaction liquid chromatography (HILIC) followed by positive-ion electrospray ionization mass spectrometry (ESI-MS) using a single internal lipid standard to which all class specific response factors (RFs) are related to. The developed method enables the nontargeted quantitation of lipid classes and molecules inside these classes in contrast to the conventional targeted quantitation, which is based on predefined selected reaction monitoring (SRM) transitions for selected lipids only. In the nontargeted quantitation method described here, concentrations of lipid classes are obtained by the peak integration in HILIC chromatograms multiplied by their RFs related to the single internal standard (i.e., sphingosyl PE, d17:1/12:0) used as common reference for all polar lipid classes. The accuracy, reproducibility and robustness of the method have been checked by various means: (1) the comparison with conventional lipidomic quantitation using SRM scans on a triple quadrupole (QqQ) mass analyzer, (2) (31)P nuclear magnetic resonance (NMR) quantitation of the total lipid extract, (3) method robustness test using subsequent measurements by three different persons, (4) method transfer to different HPLC/MS systems using different chromatographic conditions, and (5) comparison with previously published results for identical samples, especially human reference plasma from the National Institute of Standards and Technology (NIST human plasma). Results on human plasma, egg yolk and porcine liver extracts are presented and discussed.

This review summarizes the state-of-art in liquid chromatography-mass spectrometry (LC-MS) and related techniques with the main focus on recent developments in the last decade. LC-MS records an enormous growth in recent years due to the application potential in analytical chemistry, biochemistry, pharmaceutical analysis, clinical analysis and many other fields, where the qualitative and quantitative characterization of complex organic, bioorganic and organometallic mixtures is needed. Beginners and moderately experienced LC-MS users may be confused by the number of different LC-MS systems on the market, therefore an actual overview of mass spectrometers designed for the LC-MS configuration and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) from main manufacturers is compiled here together with an independent assessment of their advantages and limitations. Current trends in terms of mass analyzers, ionization techniques, fast LC-MS, LC-MALDI-MS, ion mobility spectrometry used in LC-MS, quantitation issues specific to MS and emerging mass spectrometric approaches complementary to LC-MS are discussed as well., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Biotransformation enzymes can, to a certain extent, protect parasitic worms against the toxic effects of anthelmintics and can contribute to drug-resistance development. The objective of our work was (1) to find and identify phase I and II metabolites of the anthelmintic praziquantel (PZQ) formed by the lancet fluke (Dicrocoelium dendriticum) and the rat tapeworm (Hymenolepis diminuta) and (2) to compare PZQ metabolites in helminths with PZQ biotransformation in rat as host species. Ultra high performance liquid chromatography/tandem mass spectrometry (UHPLC/MS/MS) was used for this purpose. During in vitro incubations, mitochondria-like and microsomes-like fractions (prepared from homogenates of adult worms or from rat liver homogenate) were incubated with 10 and 100 μM PZQ. Liquid/liquid extraction was used for samples during in vitro experiments. In the ex vivo study, living D. dendriticum and H. diminuta adults were incubated in RPMI-1640 medium in the presence of 50 nM or 100 nM PZQ for 24h. After incubation, the worms were removed from the medium and homogenized. Homogenates of worms, medium from the incubation of worms or rat hepatocytes and rat urine (collected during 24h after oral PZQ administration) were separately extracted using solid-phase extraction. The results showed that both D. dendriticum and H. diminuta enzymatic systems are not able to metabolize PZQ. On the other hand, thirty one different phase I and four phase II PZQ metabolites were detected in rat samples using UHPLC/MS/MS analyses. These results show that our experimental helminths, as the members of tapeworm and fluke groups of parasites, are not able to deactivate PZQ, and that the biotransformation enzymes of the studied helminths do not contribute to PZQ-resistance., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The analysis of organometallic compounds with mass spectrometry has some special features in comparison with organic and bioorganic compounds. The first step is the choice of a suitable ionization technique, where the electrospray ionization is certainly the best possibility for most classes of organometallic compounds and metal complexes. Some ionization mechanisms of organometallic compounds are comparable to organic molecules, such as protonation/deprotonation, and adduct formation with sodium or potassium ions; however, in many cases, different mechanisms and their combinations complicate the spectra interpretation. Organometallics frequently undergo various types of adduct and polymerization reactions that result in significantly higher masses observed in the spectra in comparison to molecular weights of studied compounds. Metal elements typically have more natural isotopes than common organic elements, which cause characteristic wide distributions of isotopic peaks; for example, tin has ten natural isotopes. The isotopic pattern can be used for the identification of the type and number of metal elements in particular ions. The ionization and fragmentation behavior also depend on the type of metal atom; therefore, our discussion of mass spectra interpretation is divided according to the different type of organometallic compounds. Among various types of mass spectrometers available on the market, trap-based analyzers (linear or spherical ion-traps, Orbitrap) are suitable to study complex fragmentation pathways of organometallic ions and their adducts, whereas high-resolution and high-mass accuracy analyzers (time-of-flight-based analyzers, or Fourier transform-based analyzers-Orbitrap or ion cyclotron resonance mass spectrometers) provide accurate masses applicable for the determination of the elemental composition of individual ions., (Copyright © 2010 Wiley Periodicals, Inc.)

Fatty acid (FA) and triacylglycerol (TG) composition of natural oils and fats intake in the diet has a strong influence on the human health and chronic diseases. In this work, non-aqueous reversed-phase (NARP) and silver-ion high-performance liquid chromatography with atmospheric pressure chemical ionization mass spectrometry detection and gas chromatography with flame-ionization detection (GC/FID) and mass spectrometry detection are used for the characterization of FA and TG composition in complex samples of animal fats from fallow deer, red deer, sheep, moufflon, wild boar, cock, duck and rabbit. The FA composition of samples is determined based on the GC/FID analysis of FA methyl esters. In total, 81 FAs of different acyl chain length, double bond (DB) number, branched/linear, cis-/trans- and DB positional isomers are identified. TGs in animal fats contain mainly monounsaturated and saturated FAs. High amounts of branched and trans-FAs are observed in the samples of ruminants. In NARP mode, individual TG species are separated including the separation of trans- and branched TGs. Silver-ion mode provides the separation of TG regioisomers, which enables the determination of their ratios. Great differences in the preference of unsaturated and saturated FAs in the sn-2 position on the glycerol skeleton are observed among individual animal fats. Unsaturated FAs are preferentially occupied in the sn-2 position in all animal samples except for wild boar with the strong preference of saturated FAs in the sn-2 position., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Biotransformation products of two potential antineoplastic agents, benfluron and dimefluron, are characterized using our integrated approach based on the combination of high-performance liquid chromatography (HPLC) separation of phase I and phase II metabolites followed by photodiode-array UV detection and electrospray ionization tandem mass spectrometry (MS/MS). High mass accuracy measurement allows confirmation of an elemental composition and metabolic reactions according to exact mass defects. The combination of different HPLC/MS/MS scans, such as reconstructed ion current chromatograms, constant neutral loss chromatograms or exact mass filtration, helps the unambiguous detection of low abundance metabolites. The arene oxidation, N-oxidation, N-demethylation, O-demethylation, carbonyl reduction, glucuronidation and sulfation are typical mechanisms of the metabolite formation. The interpretation of their tandem mass spectra enables the distinction of demethylation position (N- vs. O-) as well as to differentiate N-oxidation from arene oxidation for both phase I and phase II metabolites. Two metabolic pathways are rather unusual for rat samples, i.e., glucosylation and double glucuronidation. The formation of metabolites that lead to a significant change in the chromophoric system of studied compounds, such as the reduction of carbonyl group in 7H-benzo[c]fluorene-7-one chromophore, is reflected in their UV spectra, which provides valuable complementary information to MS/MS data., (Copyright © 2011 John Wiley & Sons, Ltd.)

Lipids are important components in all biological tissues having many essential roles associated with the proper function of the organism. Their analysis in the biological tissues and body fluids is a challenging task due to the extreme sample complexity of polar lipids and to their amphiphilic character. In this work, we describe a new method for the characterization of the lipid composition in various tissues, using off-line two-dimensional coupling of hydrophilic interaction liquid chromatography (HILIC) and reversed-phase (RP) high-performance liquid chromatography coupled to electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) mass spectrometry. In the first dimension the total lipid extracts are fractioned using HILIC into individual lipid classes. In total, 19 lipid classes (+3 regioisomeric pairs) that cover a wide range of polarities are separated in one analytical run, which is the highest number of analyzed lipid classes reported so far. The lysophospholipid regioisomers are also separated in HILIC mode followed by the identification based on the characteristic ESI mass spectra. The collected fractions of the various lipid classes are further separated in the RP mode, which offers an excellent resolution of the individual lipid species. Their ESI or APCI mass spectra give correct information on the fatty acid composition and on the individual regioisomeric positions on the glycerol skeleton. Off-line coupling of both modes enables the comprehensive analysis of plant and animal samples as illustrated on the analysis of egg yolk, soya and porcine brain tissues., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Silver-ion high-performance liquid chromatography (HPLC) coupled to atmospheric pressure chemical ionization mass spectrometry (APCI-MS) is used for the regioisomeric analysis of triacylglycerols (TGs). Standard mixtures of TG regioisomers are prepared by the randomization reaction from 8 mono-acid TG standards (tripalmitin, tristearin, triarachidin, triolein, trielaidin, trilinolein, trilinolenin and tri-gamma-linolenin). In total, 32 different regioisomeric doublets and 11 triplets are synthesized, separated by silver-ion HPLC using three serial coupled chromatographic columns giving a total length of 75cm. The retention of TGs increases strongly with the double bond (DB) number and slightly for regioisomers having more DBs in sn-1/3 positions. DB positional isomers (linolenic vs. γ-linolenic acids) are also separated and their reverse retention order in two different mobile phases is demonstrated. APCI mass spectra of all separated regioisomers are measured on five different mass spectrometers: single quadrupole LC/MSD (Agilent Technologies), triple quadrupole API 3000 (AB SCIEX), ion trap Esquire 3000 (Bruker Daltonics), quadrupole time-of-flight micrOTOF-Q (Bruker Daltonics) and LTQ Orbitrap XL (Thermo Fisher Scientific). The effect of different types of mass analyzer on the ratio of [M+H-R(i)COOH](+) fragment ions in APCI mass spectra is lower compared to the effect of the number of DBs, their position on the acyl chain and the regiospecific distribution of acyl chains on the glycerol skeleton. Presented data on [M+H-R(i)COOH](+) ratios measured on five different mass analyzers can be used for the direct regioisomeric determination in natural and biological samples., (Copyright © 2010 Elsevier B.V. All rights reserved.)