Search

Mass spectrometry imaging (MSI) provides insight into the molecular distribution of a broad range of compounds and, therefore, is frequently applied in the pharmaceutical industry. Pharmacokinetic and toxicological studies deploy MSI to localize potential drugs and their metabolites in biological tissues but currently require other analytical tools to quantify these pharmaceutical compounds in the same tissues. Quantitative mass spectrometry imaging (Q-MSI) is a field with challenges due to the high biological variability in samples combined with the limited sample cleanup and separation strategies available prior to MSI. In consequence, more selectivity in MSI instruments is required. This can be provided by multiple reaction monitoring (MRM) which uses specific precursor ion-product ion transitions. This targeted approach is in particular suitable for pharmaceutical compounds because their molecular identity is known prior to analysis. In this work, we compared different analytical platforms to assess the performance of MRM detection compared to other MS instruments/MS modes used in a Q-MSI workflow for two drug candidates (A and B). Limit of detection (LOD), linearity, and precision and accuracy of high and low quality control (QC) samples were compared between MS instruments/modes. MRM mode on a triple quadrupole mass spectrometer (QqQ) provided the best overall performance with the following results for compounds A and B: LOD 35.5 and 2.5 μg/g tissue, R2 0.97 and 0.98 linearity, relative standard deviation QC R2 0.86–0.98 and 0.86–0.98 linearity, relative standard deviation QC Graphical abstract

Botulinum neurotoxins (BoNTs) are the most potent toxins known and they cause the paralytic disease botulism in humans and animals. In order to diagnose botulism, active BoNT must be detected in biological material. Endopep-MS is a sensitive and selective method for serum samples, based on antibody capture, enzymatic cleavage of target peptides, and detection of cleavage products using matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS). In many cases of animal botulism, serum samples are not available or they do not contain detectable amounts of BoNT and liver sampling is an alternative for postmortem examinations. However, the Endopep-MS method is impaired by the inherent protease activity of liver samples. In the presented study, the Endopep-MS method has been successfully modified and validated for analysis of cattle, horse, and avian liver samples, introducing a combination of a salt washing step and a protease inhibitor cocktail. These modifications resulted in a substantial decrease in interfering signals and increase in BoNT-specific signals. This led to a substantial improvement in sensitivity for especially BoNT-C and C/D which are among the most prominent serotypes for animal botulism. Botulism was diagnosed with the new method in liver samples from dead cattle and birds from outbreaks in Sweden. Graphical Abstract Electronic supplementary material The online version of this article (10.1007/s00216-020-03001-z) contains supplementary material, which is available to authorized users.

Trace elements are essential for life and their concentration in cells and tissues must be tightly maintained and controlled to avoid pathological conditions. Established methods to measure the concentration of trace elements in biological matrices often provide only single element information, are time-consuming, and require special sample preparation. Therefore, the development of straightforward and rapid analytical methods for enhanced, multi-trace element determination in biological samples is an important and raising field of trace element analysis. Herein, we report on the development and validation of a reliable method based on total reflection X-ray fluorescence (TXRF) analysis to precisely quantify iron and other trace metals in a variety of biological samples, such as the liver, parenchymal and non-parenchymal liver cells, and bone marrow–derived macrophages. We show that TXRF allows fast and simple one-point calibration by addition of an internal standard and has the potential of multi-element analysis in minute sample amounts. The method was validated for iron by recovery experiments in homogenates in a wide concentration range from 1 to 1600 μg/L applying well-established graphite furnace atomic absorption spectrometry (GFAAS) as a reference method. The recovery rate of 99.93 ± 0.14% reveals the absence of systematic errors. Furthermore, the standard reference material “bovine liver” (SRM 1577c, NIST) was investigated in order to validate the method for further biometals. Quantitative recoveries (92–106%) of copper, iron, zinc, and manganese prove the suitability of the developed method. The limits of detection for the minute sample amounts are in the low picogram range., publishedVersion

Polyunsaturated fatty acids (PUFAs) and eicosanoids are important mediators of inflammation. The functional role of eicosanoids in metabolic-syndrome-related diseases has been extensively studied. However, their role in neuroinflammation and the development of neurodegenerative diseases is still unclear. The aim of this study was the development of a sample pretreatment protocol for the simultaneous analysis of PUFAs and eicosanoids in mouse liver and brain. Liver and brain samples of male wild-type C57BL/6J mice (11–122 mg) were used to investigate conditions for tissue rinsing, homogenization, extraction, and storage. A targeted liquid chromatography–negative electrospray ionization tandem mass spectrometry method was applied to quantify 7 PUFAs and 94 eicosanoids. The final pretreatment protocol consisted of a 5-min homogenization step by sonication in 650 μL n-hexane/2-propanol (60:40 v/v) containing 2,6-di-tert-butyl-4-methylphenol at 50 μg/mL. Homogenates representing 1 mg tissue were extracted in a single step with n-hexane/2-propanol (60:40 v/v) containing 0.1% formic acid. Autoxidation was prevented by addition of 2,6-di-tert-butyl-4-methylphenol at 50 μg/mL and keeping the samples at 4 °C during sample preparation. Extracts were dried under nitrogen and reconstituted in liquid chromatography eluent before analysis. Recovery was determined to range from 45% to 149% for both liver and brain tissue. Within-run and between-run variability ranged between 7% and 18% for PUFAs and between 1% and 24% for eicosanoids. In liver, 7 PUFAs and 15 eicosanoids were quantified; in brain, 6 PUFAs and 21 eicosanoids had significant differences within the brain substructures. In conclusion, a robust and reproducible sample preparation protocol for the multiplexed analysis of PUFAs and eicosanoids by liquid chromatography–tandem mass spectrometry in liver and discrete brain substructures was developed. Electronic supplementary material The online version of this article (10.1007/s00216-019-02170-w) contains supplementary material, which is available to authorized users.

Diacyl glycerophospholipids (GPs) belong to the most abundant lipid species in living organisms and consist of a glycerol backbone with fatty acyl groups in sn-1 and sn-2 and a polar head group in the sn-3 position. Regioisomeric mixed diacyl GPs have the same fatty acyl composition but differ in their allocation to sn-1 or sn-2 of the glycerol unit. In-depth analysis of regioisomeric mixed diacyl GP species composed of fatty acyl moieties that are similar in length and degree of saturation typically requires either chemical derivatization or sophisticated analytical instrumentation, since these types of regioisomers are not well resolved under standard ultra-performance liquid chromatography (UPLC) conditions. Here, we introduce a simple and fast method for diacyl GP regioisomer analysis employing UPLC tandem mass spectrometry (MS/MS). This GP regioisomer analysis is based both on minor chromatographic retention time shifts and on major differences in relative abundances of the two fatty acyl anion fragments observed in MS/MS. To monitor these differences with optimal precision, MS/MS spectra are recorded continuously over the UPLC elution profile of the lipid species of interest. Quantification of relative abundances of the regioisomers was performed by algorithms that we have developed for this purpose. The method was applied to commercially available mixed diacyl GP standards and to total lipid extracts of Escherichia coli (E. coli) and bovine liver. To validate our results, we determined regioisomeric ratios of phosphatidylcholine (PC) standards using phospholipase A2-specific release of fatty acids from the sn-2 position of the glycerol backbone. Our results show that most analyzed mixed diacyl GPs of biological origin exhibit significantly higher regioisomeric purity than synthetic lipid standards. In summary, this method can be implemented in routine LC-MS/MS-based lipidomics workflows without the necessity for additional chemical additives, derivatizations, or instrumentation. Electronic supplementary material The online version of this article (10.1007/s00216-018-1517-5) contains supplementary material, which is available to authorized users.

Clinical failure of novel drugs is often related to their rapid metabolism and excretion. This highlights the importance of elucidation of their pharmacokinetic profile already at the preclinical stage of drug development. Triapine, the most prominent representative of α-N-heterocyclic thiosemicarbazones, was investigated in more than 30 clinical phase I/II trials, but the results against solid tumors were disappointing. Recent investigations from our group suggested that this is, at least partially, based on the fast metabolism and excretion. In order to establish more detailed structure/activity/metabolism relationships, herein a panel of 10 different Triapine derivatives was investigated for their metabolic pathways. From the biological point of view, the panel consists of terminally dimethylated thiosemicarbazones with nanomolar IC50 values, derivatives with micromolar cytotoxicities comparable to Triapine and a completely inactive representative. To study the oxidative metabolism, a purely instrumental approach based on electrochemistry/mass spectrometry was applied and the results were compared to the data obtained from microsomal incubations. Overall, the investigated thiosemicarbazones underwent the phase I metabolic reactions dehydrogenation, hydroxylation, oxidative desulfuration (to semicarbazone and amidrazone) and demethylation. Notably, dehydrogenation resulted in a ring-closure reaction with formation of thiadiazoles. Although strong differences between the metabolic pathways of the different thiosemicarbazones were observed, they could not be directly correlated to their cytotoxicities. Finally, the metabolic pathways for the most cytotoxic compound were elucidated also in tissues collected from drug-treated mice, confirming the data obtained by electrochemical oxidation and microsomes. In addition, the in vivo experiments revealed a very fast metabolism and excretion of the compound. Graphical abstractStructure/activity/metabolisation relationships for 10 anticancer thiosemicarbazones were established using electrochemical oxidation coupled to mass spectrometry (EC-MS) and human liver microsomes analyzed by LC-MS Electronic supplementary material The online version of this article (10.1007/s00216-018-0889-x) contains supplementary material, which is available to authorized users.

The heritability of chronic hepatitis in the Labrador Retriever is studied with the aim of identifying the related gene mutation. Identification of cases and controls is largely based on instrumental neutron activation analysis (INAA) Cu determination in liver biopsies. The burden for these companion animals may be reduced if nail clippings and hair (fur) could serve as a noninvasive indicator for the hepatic Cu concentrations. No correlation was found between hepatic Cu concentrations and Cu concentrations in hair and nail samples. However, hair and nail samples were also analyzed by X-ray tube excitation, taking advantage of the X-ray Compton, Rayleigh, and Raman scattering which reflects the organic components such as the type of melanin. Principal component analysis provided first indications that some differentiation between healthy and sick dogs could indeed be obtained from hair and nail analysis. Figure Principal component analysis of scattered region of x-ray fluorescence spectra of Labrador dog nails, demonstrating the differentiation towards dogs with high and low Cu liver levels (respectively positive and negative PC2 values) reflecting hepatitis, as well as gender (PC1: negative values for female and positive values for males)

This paper describes a fast, simple and novel extraction method for total selenium and selenium species determination in food samples. Parameters influencing extraction, such as sonication time, extracting media, temperature, sample mass, ultrasound amplitude and sample/enzyme mass ratio were investigated. The enzymatic hydrolysis proposed, enhanced by probe sonication, allowed the quantitative extraction of selenium in chicken muscle, liver, kidney and feed (97, 93, 95 and 102%, respectively) in 2 min, maintaining the original Se-species integrity. Total Se content of the samples was determined using inductively coupled plasma mass spectrometry. Se-species were identified and quantified using high-performance liquid chromatography in conjunction with inductively coupled plasma mass spectrometry. Chromatographic analyses were carried out under two chromatographic conditions and led to the identification of SeMet in all samples. The accuracy of the proposed method was assessed using certified reference materials as well as microwave digestion. Potential advantages of the proposed method over traditional hydrolysis are speed, simplicity and safety of the procedure.

This paper reports the identification and quantification of superoxide dismutase in the liver of Mus musculus and Mus spretus mice using a metallomics analytical approach. The approach consisted of using orthogonal chromatographic systems coupled to ICP-MS and UV detectors. Size-exclusion fractionation of the cytosolic extracts was followed by anion-exchange chromatographic separation of Cu- and Zn-containing species. After purification then tryptic digestion, Cu- and Zn-containing superoxide dismutase was identified by nESI-QqTOF. The MS-MS spectra of doubly charged peptides, with the Mascot searching engine, were used to obtain the sequence of the protein.

Despite CYP induction in vitro in precision-cut liver slices (LS) is well documented, there are no standardised assays for determining CYP activity as a major end-point. In this paper, short-term assays with intact and homogenised LS from male and female rats were directly compared. We obtained similar results for 7-ethoxycoumarine O-deethylation (ECOD) with LS from both sexes: higher basal activities were measured in LS homogenate, whereas slightly stronger induction by BNF was found with intact LS. CYP3A-dependent basal and dexamethasone (Dex)-induced 2beta-, 15beta- and 6beta-testosterone hydroxylation (TH) rates were higher in both intact and homogenised LS from male compared to female rats. CYP3A induction in vitro could likewise be detected in intact and homogenised LS preferentially by determining 2beta- and 15beta-TH, with higher induction factors observed in LS from females. 6beta-TH seems to be less inducible in intact LS of males. In vivo pretreatment of liver donors with BNF and Dex did not substantially disturb the subsequent in vitro induction of ECOD and TH, respectively.

This paper reports characterization of the behavior of five pneumatic micronebulizers based on slightly different designs in inductively coupled plasma atomic-emission spectrometry and mass spectrometry (ICP–AES and ICP–MS). Two nebulizers were used as reference nebulizers, a high-efficiency nebulizer (HEN) and a micromist (MM). They were compared with a commercially available PFA (tetrafluoroethylene–per- fluoroalkyl vinyl ether copolymer) nebulizer and with two new prototypes called the polymeric pneumatic concentric nebulizer (PMN) and the high-solids micronebulizer (HSM). The dimensions of the nebulizers, the gas back-pressure, and the free liquid uptake rates were measured. The study also included tertiary aerosol dropsize distributions, analyte transport rate, and analytical figures of merit, i.e. sensitivities and limits of detection, both in ICP–AES and ICP–MS. Recoveries for two food solid reference materials were also determined. Overall, the results indicated that the PFA and the HEN nebulizers provided the best results. These two nebulizers delivered a higher mass of analyte to the plasma and showed better sensitivies giving lower limits of detection than the PMN, HSM and MM. The results revealed that the liquid prefilming effect occurring before aerosol production in the PFA nebulizer promoted more effi- cient interaction of liquid and gas, thus affording good results even though gas back-pressure values could be maintained below 3 bar. In contrast, the HEN had to be operated at about 7 bar under the same conditions. Nebulizer design did not have a relevant effect on the recovery, which confirmed that the spray chamber plays an important role in terms of non-spectroscopic interferences.