Advances in tissue section preparation for MALDI imaging MS

Enriched by a decade of remarkable developments, matrix-assisted laser desorption ionization imaging mass spectrometry (MALDI IMS) has witnessed a phenomenal expansion. Initially introduced for the mapping of peptides and intact proteins from mammalian tissue sections, MALDI IMS applications now extend to a wide range of molecules including peptides, lipids, metabolites and xenobiotics. Technology and methodology are quickly evolving to push the limits of the technique forward. Within a short period of time, numerous protocols and concepts have been developed and introduced in tissue section preparation, nonexhaustively including in situ tissue chemistries and solvent-free matrix depositions. Considering the past progress and current capabilities, this Review aims to cover the different aspects and challenges of tissue section preparation for MALDI IMS.

(MA)LDI MS Imaging at High Specificity and Sensitivity

(Matrix-assisted) laser desorption/ionization ((MA)LDI) mass spectrometry imaging (MSI) has been driven by remarkable technological developments in the last couple of years. Although molecular information of a wide range of molecules including peptides, lipids, metabolites, and xenobiotics can be mapped, (MA)LDI MSI only leads to the detection of the most abundant soluble molecules in the cells and, consequently, does not provide access to the least expressed species, which can be very informative in the scope of disease research. Within a short period of time, numerous protocols and concepts have been developed and introduced in order to increase MSI sensitivity, including in situ tissue chemistry and solvent-free matrix depositions. In this chapter, we will discuss some of the latest developments in the field of high-sensitivity MSI using solvent-free matrix depositions and will detail protocols of two methods with their capability of enriching molecular MSI signal as demonstrated within our laboratory.

Separation of small metabolites and lipids in spectra from biopsies by diffusion-weighted HR-MAS NMR: a feasibility study.

High Resolution Magic Angle Spinning (HR-MAS) NMR allows metabolic characterization of biopsies. HR-MAS spectra from tissues of most organs show strong lipid contributions that are overlapping metabolite regions, which hamper metabolite estimation. Metabolite quantification and analysis would benefit from a separation of lipids and small metabolites. Generally, a relaxation filter is used to reduce lipid contributions. However, the strong relaxation filter required to eliminate most of the lipids also reduces the signals for small metabolites. The aim of our study was therefore to investigate different diffusion editing techniques in order to employ diffusion differences for separating lipid and small metabolite contributions in the spectra from different organs for unbiased metabonomic analysis. Thus, 1D and 2D diffusion measurements were performed, and pure lipid spectra that were obtained at strong diffusion weighting (DW) were subtracted from those obtained at low DW, which include both small metabolites and lipids. This subtraction yielded almost lipid free small metabolite spectra from muscle tissue. Further improved separation was obtained by combining a 1D diffusion sequence with a T2-filter, with the subtraction method eliminating residual lipids from the spectra. Similar results obtained for biopsies of different organs suggest that this method is applicable in various tissue types. The elimination of lipids from HR-MAS spectra and the resulting less biased assessment of small metabolites have potential to remove ambiguities in the interpretation of metabonomic results. This is demonstrated in a reproducibility study on biopsies from human muscle.

Liquid chromatography-tandem mass spectrometry (LC/APCI-MS/MS) methods for the quantification of captan and folpet phthalimide metabolites in human plasma and urine.

Captan and folpet are fungicides largely used in agriculture. They have similar chemical structures, except that folpet has an aromatic ring unlike captan. Their half-lives in blood are very short, given that they are readily broken down to tetrahydrophthalimide (THPI) and phthalimide (PI), respectively. Few authors measured these biomarkers in plasma or urine, and analysis was conducted either by gas chromatography coupled to mass spectrometry or liquid chromatography with UV detection. The objective of this study was thus to develop simple, sensitive and specific liquid chromatography-atmospheric pressure chemical ionization-tandem mass spectrometry (LC/APCI-MS/MS) methods to quantify both THPI and PI in human plasma and urine. Briefly, deuterated THPI was added as an internal standard and purification was performed by solid-phase extraction followed by LC/APCI-MS/MS analysis in negative ion mode for both compounds. Validation of the methods was conducted using spiked blank plasma and urine samples at concentrations ranging from 1 to 250 μg/L and 1 to 50 μg/L, respectively, along with samples of volunteers and workers exposed to captan or folpet. The methods showed a good linearity (R (2) > 0.99), recovery (on average 90% for THPI and 75% for PI), intra- and inter-day precision (RSD, <15%) and accuracy (<20%), and stability. The limit of detection was 0.58 μg/L in urine and 1.47 μg/L in plasma for THPI and 1.14 and 2.17 μg/L, respectively, for PI. The described methods proved to be accurate and suitable to determine the toxicokinetics of both metabolites in human plasma and urine.

Simultaneous LC-MS/MS quantification of P-glycoprotein and cytochrome P450 probe substrates and their metabolites in DBS and plasma.

BACKGROUND: An LC-MS/MS method has been developed for the simultaneous quantification of P-glycoprotein (P-gp) and cytochrome P450 (CYP) probe substrates and their Phase I metabolites in DBS and plasma. P-gp (fexofenadine) and CYP-specific substrates (caffeine for CYP1A2, bupropion for CYP2B6, flurbiprofen for CYP2C9, omeprazole for CYP2C19, dextromethorphan for CYP2D6 and midazolam for CYP3A4) and their metabolites were extracted from DBS (10 µl) using methanol. Analytes were separated on a reversed-phase LC column followed by SRM detection within a 6 min run time. RESULTS: The method was fully validated over the expected clinical concentration range for all substances tested, in both DBS and plasma. The method has been successfully applied to a PK study where healthy male volunteers received a low dose cocktail of the here described P-gp and CYP probes. Good correlation was observed between capillary DBS and venous plasma drug concentrations. CONCLUSION: Due to its low-invasiveness, simple sample collection and minimal sample preparation, DBS represents a suitable method to simultaneously monitor in vivo activities of P-gp and CYP.

Plasma levels of trimipramine and metabolites in four patients: determination of the enantiomer concentrations of the hydroxy metabolites.

A two-step high-performance liquid chromatography method is described, using a CN column and an alpha 1-acid glycoprotein column, which allows the measurement of the enantiomers of the hydroxy metabolites of trimipramine in plasma of trimipramine-treated patients. Of the four patients analyzed, three showed approximately equimolar concentrations of the (D)- and (L)-enantiomers of the hydroxy metabolites (2-hydroxy-trimipramine and 2-hydroxy desmethyltrimipramine), and one was found to have roughly twice as much of the (L)-form and of the (D)-form of 2-hydroxy trimipramine and 2-hydroxy desmethyltrimipramine. From the data available on the pharmacological effects of the enantiomers of trimipramine, it is postulated that this interindividual variability in its pharmacokinetics is another factor that could contribute to the interindividual variability in its pharmacodynamics.

Determination of human plasma levels of levo-alpha-acetylmethadol and its metabolites by gas chromatography-mass spectrometry.

A gas chromatography-mass spectrometry (GC-MS) method is presented which allows the simultaneous determination of the plasma concentrations of the levo-alpha-acetylmethadol (LAAM) and of its active metabolites (NorLAAM and DiNorLAAM), after derivatization with the reagent trifluoroacetic anhydride (TFAA). No interferences from endogenous compounds were observed following the extraction of plasma samples from 11 different human subjects. The standard curves were linear over a working range of 5-200ng/ml for the three compounds. Recoveries measured at three concentrations ranged from 47 to 67% for LAAM, from 50 to 69% for NorLAAM and from 28 to 50% for DiNorLAAM. Intra- and interday coefficients of variation determined at three concentrations ranged from 5 to 13% for LAAM, from 3 to 9% for NorLAAM and from 5 to 13% for DiNorLAAM. The limits of quantitation of the method were found to be 4ng/ml for the three compounds. No interference was noted from methadone. This sensitive and specific analytical method could be useful for assessing the in vivo relationship between LAAM's blood levels, clinical efficacy and/or cardiotoxicity

Detection of nandrolone metabolites in urine after a football game in professional and amateur players: a Bayesian comparison.

Nandrolone (19-nortestosterone) is a widely used anabolic steroid in sports where strength plays an essential role. Once nandrolone has been metabolised, two major metabolites are excreted in urine, 19-norandrosterone (NA) and 19-noretiocholanolone (NE). In 1997, in France, quite a few sportsmen had concentrations of 19-norandrosterone very close to the IOC cut off limit (2ng/ml). At that time, a debate took place about the capability of the human male body to produce by itself these metabolites without any intake of nandrolone or related compounds. The International Football Federation (FIFA) was very concerned with this problematic, especially because the World Cup was about to start in France. In this respect, a statistical study was held with all football players from the first and second divisions of the Swiss Football National League. All players gave a urine sample after effort and around 6% of them showed traces of 19-norandrosterone. These results were compared with amateur football players (control group) and around 6% of them had very small amounts of 19-norandrosterone and/or 19-noretiocholanolone in urine after effort, whereas none of them had detectable traces of one or the other metabolite before effort. The origin of these compounds in urine after a strenuous physical activity is still unknown, but three hypotheses can be put forward. First, an endogenous production of nandrolone metabolites takes place. Second, nandrolone metabolites are released from the fatty tissues after an intake of nandrolone, some related compounds or some contaminated nutritive supplements. Finally, the sportsmen may have taken something during or just before the football game.

A single LC-tandem mass spectrometry method for the simultaneous determination of 14 antimalarial drugs and their metabolites in human plasma.

Among the various determinants of treatment response, the achievement of sufficient blood levels is essential for curing malaria. For helping us at improving our current understanding of antimalarial drugs pharmacokinetics, efficacy and toxicity, we have developed a liquid chromatography-tandem mass spectrometry method (LC-MS/MS) requiring 200mul of plasma for the simultaneous determination of 14 antimalarial drugs and their metabolites which are the components of the current first-line combination treatments for malaria (artemether, artesunate, dihydroartemisinin, amodiaquine, N-desethyl-amodiaquine, lumefantrine, desbutyl-lumefantrine, piperaquine, pyronaridine, mefloquine, chloroquine, quinine, pyrimethamine and sulfadoxine). Plasma is purified by a combination of protein precipitation, evaporation and reconstitution in methanol/ammonium formate 20mM (pH 4.0) 1:1. Reverse-phase chromatographic separation of antimalarial drugs is obtained using a gradient elution of 20mM ammonium formate and acetonitrile both containing 0.5% formic acid, followed by rinsing and re-equilibration to the initial solvent composition up to 21min. Analyte quantification, using matrix-matched calibration samples, is performed by electro-spray ionization-triple quadrupole mass spectrometry by selected reaction monitoring detection in the positive mode. The method was validated according to FDA recommendations, including assessment of extraction yield, matrix effect variability, overall process efficiency, standard addition experiments as well as antimalarials short- and long-term stability in plasma. The reactivity of endoperoxide-containing antimalarials in the presence of hemolysis was tested both in vitro and on malaria patients samples. With this method, signal intensity of artemisinin decreased by about 20% in the presence of 0.2% hemolysed red-blood cells in plasma, whereas its derivatives were essentially not affected. The method is precise (inter-day CV%: 3.1-12.6%) and sensitive (lower limits of quantification 0.15-3.0 and 0.75-5ng/ml for basic/neutral antimalarials and artemisinin derivatives, respectively). This is the first broad-range LC-MS/MS assay covering the currently in-use antimalarials. It is an improvement over previous methods in terms of convenience (a single extraction procedure for 14 major antimalarials and metabolites reducing significantly the analytical time), sensitivity, selectivity and throughput. While its main limitation is investment costs for the equipment, plasma samples can be collected in the field and kept at 4 degrees C for up to 48h before storage at -80 degrees C. It is suited to detecting the presence of drug in subjects for screening purposes and quantifying drug exposure after treatment. It may contribute to filling the current knowledge gaps in the pharmacokinetics/pharmacodynamics relationships of antimalarials and better define the therapeutic dose ranges in different patient populations.

Characterization of metabolites in infiltrating gliomas using ex vivo &supl;H high-resolution magic angle spinning spectroscopy.

Gliomas are routinely graded according to histopathological criteria established by the World Health Organization. Although this classification can be used to understand some of the variance in the clinical outcome of patients, there is still substantial heterogeneity within and between lesions of the same grade. This study evaluated image-guided tissue samples acquired from a large cohort of patients presenting with either new or recurrent gliomas of grades II-IV using ex vivo proton high-resolution magic angle spinning spectroscopy. The quantification of metabolite levels revealed several discrete profiles associated with primary glioma subtypes, as well as secondary subtypes that had undergone transformation to a higher grade at the time of recurrence. Statistical modeling further demonstrated that these metabolomic profiles could be differentially classified with respect to pathological grading and inter-grade conversions. Importantly, the myo-inositol to total choline index allowed for a separation of recurrent low-grade gliomas on different pathological trajectories, the heightened ratio of phosphocholine to glycerophosphocholine uniformly characterized several forms of glioblastoma multiforme, and the onco-metabolite D-2-hydroxyglutarate was shown to help distinguish secondary from primary grade IV glioma, as well as grade II and III from grade IV glioma. These data provide evidence that metabolite levels are of interest in the assessment of both intra-grade and intra-lesional malignancy. Such information could be used to enhance the diagnostic specificity of in vivo spectroscopy and to aid in the selection of the most appropriate therapy for individual patients.

Untargeted profiling of urinary steroid metabolites after testosterone ingestion: opening new perspectives for antidoping testing.

AIM: Antidoping procedures are expected to greatly benefit from untargeted metabolomic approaches through the discovery of new biomarkers of prohibited substances abuse. RESULTS: Endogenous steroid metabolites were monitored in urine samples from a controlled elimination study of testosterone undecanoate after ingestion. A platform coupling ultra-high pressure LC with high-resolution quadrupole TOF MS was used and high between-subject metabolic variability was successfully handled using a multiblock data analysis strategy. Links between specific subsets of metabolites and influential genetic polymorphisms of the UGT2B17 enzyme were highlighted. CONCLUSION: This exploratory metabolomic strategy constitutes a first step toward a better understanding of the underlying patterns driving the high interindividual variability of steroid metabolism. Promising biomarkers were selected for further targeted study.

Determination of nicotine and nicotine metabolites in urine by hydrophilic interaction chromatography-tandem mass spectrometry: Potential use of smokeless tobacco products by ice hockey players.

Consumption of nicotine in the form of smokeless tobacco (snus, snuff, chewing tobacco) or nicotine-containing medication (gum, patch) may benefit sport practice. Indeed, use of snus seems to be a growing trend and investigating nicotine consumption amongst professional athletes is of major interest to sport authorities. Thus, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the detection and quantification of nicotine and its principal metabolites cotinine, trans-3-hydroxycotinine, nicotine-N'-oxide and cotinine-N-oxide in urine was developed. Sample preparation was performed by liquid-liquid extraction followed by hydrophilic interaction chromatography-tandem mass spectrometry (HILIC-MS/MS) operated in electrospray positive ionization (ESI) mode with selective reaction monitoring (SRM) data acquisition. The method was validated and calibration curves were linear over the selected concentration ranges of 10-10,000 ng/mL for nicotine, cotinine, trans-3-hydroxycotinine and 10-5000 ng/mL for nicotine-N'-oxide and cotinine-N-oxide, with calculated coefficients of determination (R(2)) greater than 0.95. The total extraction efficiency (%) was concentration dependent and ranged between 70.4 and 100.4%. The lower limit of quantification (LLOQ) for all analytes was 10 ng/mL. Repeatability and intermediate precision were ?9.4 and ?9.9%, respectively. In order to measure the prevalence of nicotine exposure during the 2009 Ice Hockey World Championships, 72 samples were collected and analyzed after the minimum of 3 months storage period and complete removal of identification means as required by the 2009 International Standards for Laboratories (ISL). Nicotine and/or metabolites were detected in every urine sample, while concentration measurements indicated an exposure within the last 3 days for eight specimens out of ten. Concentrations of nicotine, cotinine, trans-3-hydroxycotinine, nicotine-N'-oxide and cotinine-N-oxide were found to range between 11 and 19,750, 13 and 10,475, 10 and 8217, 11 and 3396, and 13 and 1640 ng/mL, respectively. When proposing conservative concentration limits for nicotine consumption prior and/or during the games (50 ng/mL for nicotine, cotinine and trans-3-hydroxycotinine and 25 ng/mL for nicotine-N'-oxide and cotinine-N-oxide), about half of the hockey players were qualified as consumers. These findings significantly support the likelihood of extensive smokeless nicotine consumption. However, since such conclusions can only be hypothesized, the potential use of smokeless tobacco as a doping agent in ice hockey requires further investigation.

Dietary lipids reduce the expression of carotenoid-based coloration in Lacerta vivipara

1. The importance of dietary lipids for carotenoid-based ornaments has rarely been investigated, although theory predicts that dietary lipids may control the development of these widespread animal signals. Dietary lipids have been suggested to enhance the expression of male carotenoid-based ornaments because they provide carotenoids with a hydrophobic domain that facilitates their absorption and transport. Dietary lipids may also enhance the uptake of tocopherols (vitamin E), which share common absorption and transport routes with carotenoids. Here, we test whether dietary lipids enhance carotenoid availability and male carotenoid-based colorations. We also explore the effects of dietary lipids on plasma tocopherol concentration, which allow disentangling between different pathways that may explain how dietary lipids affect ornamental expression. 2. Following a two-factorial design, we manipulated dietary access of naturally occurring fatty acids (oleic acid) and carotenoids (lutein and zeaxanthin) and measured its effects on the circulating concentrations of carotenoids (lutein and zeaxanthin) and vitamin E (α- and γ-(β-) tocopherols) and on the ventral, carotenoid-based coloration of male common lizards (Lacerta vivipara). 3. Lutein but not zeaxanthin plasma concentrations increased with carotenoid supplementation, which, however, did not affect coloration. Lipid intake negatively affected circulating concentrations of lutein and γ-(β-) tocopherol and led to significantly less orange colorations. The path analysis suggests that a relationship between the observed colour change and the change in plasma concentrations of γ-(β-) tocopherol may exist. 4. Our study shows for the first time that dietary lipids do not enhance but reduce the intensity of male carotenoid-based ornaments. Although dietary lipids affected plasma carotenoid concentration, its negative effect on coloration appeared to be linked to lower vitamin E plasma concentrations. These findings suggest that a conflict between dietary lipids and carotenoid and tocopherol uptake may arise if these nutrients are independently obtained from natural diets and that such conflict may reinforce signal honesty in carotenoid-based ornaments. They also suggest that, at least in the common lizard, sexual selection with respect to carotenoid-based coloration may select for males with low antioxidant capacity and thus for males of superior health.