A multivariate data analysis approach for the investigation of in vitro derived metabolites of ACP-105 in comparison with human in vivo metabolites.

Selective androgen receptor modulators (SARMs) such as ACP-105 are prohibited in sports due to their anabolic properties. ACP-105 has in previous equine studies shown to undergo extensive metabolism, which makes its metabolite profile important to investigate in humans, since the metabolism is unknown in this species. The aims of the study were to systematically optimize in vitro microsome incubations for improved metabolite yield and to utilize a multivariate data analysis (MVDA) approach to aid the metabolite discovery. Microsomes together with S9 fractions were used at optimal conditions, both with and without phase II additives. Furthermore, the relevance of the in vitro derived metabolites was evaluated as analytical targets in doping control by comparison with results from a human post-administration urine sample collected after a single dose of 100 µg ACP-105. All samples were analyzed with liquid chromatography - Orbitrap mass spectrometry. The use of the systematical optimization and MVDA greatly simplified the search and a total of 18 in vitro metabolites were tentatively identified. The yield of the two main monohydroxylated isomers increased by 24 and 10 times, respectively. In the human urine sample, a total of seven metabolites of ACP-105, formed by a combination of hydroxylations and glucuronic acid conjugations, were tentatively identified. The main metabolites were two monohydroxylated forms that are suggested as analytical targets for human doping control after hydrolysis. All the in vivo metabolites could be detected with the MVDA approach on the in vitro models, demonstrating its usefulness for prediction of the in vivo metabolite profile.

Equine in vivo metabolite profiling of the selective androgen receptor modulator LGD-3303 for doping control.

LGD-3303 is a Selective Androgen Receptor Modulator (SARM) that is prohibited in both equine and human sports due to its anabolic properties. The aim of this study was to investigate the equine in vivo metabolite profile of LGD-3303 and identify drug metabolites that can be suitable as new and improved analytical targets for equine doping control. This was performed by an oral administration of 0.05 mg·kg-1 LGD-3303 to horses, where blood and urine samples were collected up to 96 h after administration. The in vivo samples consisting of plasma, urine and hydrolyzed urine were analyzed utilizing ultra-high performance liquid chromatography hyphenated to a Q Exactive™ Orbitrap™ high resolution mass spectrometer with a heated electrospray ionization source. A total of eight metabolites of LGD-3303 were tentatively identified, including one carboxylated and several hydroxylated metabolites in combination with glucuronic acid conjugates. A monohydroxylated metabolite is suggested as an analytical target for doping control analysis of plasma and urine after hydrolysis with ß-glucuronidase, due to the high intensity and prolonged detection time in comparison to parent LGD-3303.

Investigation of neomycin biodegradation conditions using ericoid mycorrhizal and white rot fungal species.

BACKGROUND: In the search for methods to biodegrade recalcitrant compounds, the use of saprotrophic fungi and white rot fungi, in particular belonging to the phylum Basidiomycota, has gained interest. This group of fungi possesses a battery of unspecific extracellular enzymes that can be utilized in the biodegradation of preferably phenolic compounds. In this work, it was investigated under which conditions the white rot fungus Trametes versicolor and the ericoid mycorrhizal fungus Rhizoscyphus ericae (belonging to the phylum Ascomycota) could be used to biodegrade the antibiotic aminoglycoside neomycin at co-metabolic conditions in which external nutrients were supplied. Furthermore, it was also investigated whether a biodegradation could be accomplished using neomycin as the sole nutrient. RESULTS: The results show that both species can biodegrade neomycin 70% under co-metabolic conditions during a one-week time course and that Rhizoscyphus ericae is able to use neomycin as sole nutrient and to approximatively biodegrade it 60% under chosen non co-metabolic conditions. At selected conditions, the biodegradation of neomycin using Rhizoscyphus ericae was monitored by oxidation products of D-ribose which is a hydrolysis product of neomycin. CONCLUSION: The results are of general interest in the search for fungal species that can biodegrade recalcitrant compounds without the need of external nutrients. The key future application area that will be investigated is purification of waste from recombinant protein production in which neomycin, nutrients and E. coli with neomycin resistance genes are present.

Neomycin removal using the white rot fungus Trametes versicolor.

The presence of antibiotic resistance genes in wastewater treatment plants (WWTPs), and in river and lake recipients show the need to develop new antibiotic removal strategies. The aminoglycoside antibiotic class is of special concern since the chemical structure of these compounds limits the choices of removal technologies. The experimental design included fungal mediated in vivo and in vitro experiments. The experiments were performed in Erlenmeyer flasks under non-sterile conditions. In the study, the role of the laccase redox mediator 4-hydroxy benzoic acid (HBA) in the removal of neomycin was investigated. The specific objective of the study was to conclude whether it is possible to use the white rot fungus (WRF) Trametes versicolor to biodegrade neomycin. It was shown that it is feasible to remove 34% neomycin in vitro (excluding living fungal cells) by laccase-HBA mediated extracellular biodegradation. In the in vivo experiments, polyurethane foam (PUF) was used as supporting material to immobilize fungal mycelia on. The presence of living fungal cells facilitated a removal of approximately 80% neomycin in the absence of HBA. Using liquid chromatography-high resolution-mass spectrometry, it was possible to tentatively identify oxidation products of neomycin hydrolysates. The results in this study open up the possibility to implement a pretreatment plant (PTP) aimed for neomycin removal.

Oceanographic setting influences the prokaryotic community and metabolome in deep-sea sponges.

Marine sponges (phylum Porifera) are leading organisms for the discovery of bioactive compounds from nature. Their often rich and species-specific microbiota is hypothesised to be producing many of these compounds. Yet, environmental influences on the sponge-associated microbiota and bioactive compound production remain elusive. Here, we investigated the changes of microbiota and metabolomes in sponges along a depth range of 1232 m. Using 16S rRNA gene amplicon sequencing and untargeted metabolomics, we assessed prokaryotic and chemical diversities in three deep-sea sponge species: Geodia barretti, Stryphnus fortis, and Weberella bursa. Both prokaryotic communities and metabolome varied significantly with depth, which we hypothesized to be the effect of different water masses. Up to 35.5% of microbial ASVs (amplicon sequence variants) showed significant changes with depth while phylum-level composition of host microbiome remained unchanged. The metabolome varied with depth, with relative quantities of known bioactive compounds increasing or decreasing strongly. Other metabolites varying with depth were compatible solutes regulating osmolarity of the cells. Correlations between prokaryotic community and the bioactive compounds in G. barretti suggested members of Acidobacteria, Proteobacteria, Chloroflexi, or an unclassified prokaryote as potential producers.

Comprehensive Peak Characterization (CPC) in Untargeted LC-MS Analysis.

LC-MS-based untargeted metabolomics is heavily dependent on algorithms for automated peak detection and data preprocessing due to the complexity and size of the raw data generated. These algorithms are generally designed to be as inclusive as possible in order to minimize the number of missed peaks. This is known to result in an abundance of false positive peaks that further complicate downstream data processing and analysis. As a consequence, considerable effort is spent identifying features of interest that might represent peak detection artifacts. Here, we present the CPC algorithm, which allows automated characterization of detected peaks with subsequent filtering of low quality peaks using quality criteria familiar to analytical chemists. We provide a thorough description of the methods in addition to applying the algorithms to authentic metabolomics data. In the example presented, the algorithm removed about 35% of the peaks detected by XCMS, a majority of which exhibited a low signal-to-noise ratio. The algorithm is made available as an R-package and can be fully integrated into a standard XCMS workflow.

Automated Sequential Analysis of Hydrophilic and Lipophilic Fractions of Biological Samples: Increasing Single-Injection Chemical Coverage in Untargeted Metabolomics.

In order to increase metabolite coverage in LC-MS-based untargeted metabolomics, HILIC- and RPLC-mode separations are often combined. Unfortunately, these two techniques pose opposite requirements on sample composition, necessitating either dual sample preparations, increasing needed sample volume, or manipulation of the samples after the first analysis, potentially leading to loss of analytes. When sample material is precious, the number of analyses that can be performed is limited. To that end, an automated single-injection LC-MS method for sequential analysis of both the hydrophilic and lipophilic fractions of biological samples is described. Early eluting compounds in a HILIC separation are collected on a trap column and subsequently analyzed in the RPLC mode. The instrument configuration, composed of commercially available components, allows easy modulation of the dilution ratio of the collected effluent, with sufficient dilution to obtain peak compression in the RPLC column. Furthermore, the method is validated and shown to be fit for purpose for application in untargeted metabolomics. Repeatability in both retention times and peak areas was excellent across over 140 injections of protein-precipitated blood plasma. Finally, the method has been applied to the analysis of real perilymph samples collected in a guinea pig model. The QC sample injections clustered tightly in the PCA scores plot and showed a high repeatability in both retention times and peak areas for selected compounds.

The Effects of Sampling and Storage Conditions on the Metabolite Profile of the Marine Sponge Geodia barretti.

Geodia barretti is a deep-sea marine sponge common in the north Atlantic and waters outside of Norway and Sweden. The sampling and subsequent treatment as well as storage of sponges for metabolomics analyses can be performed in different ways, the most commonly used being freezing (directly upon collection or later) or by storage in solvent, commonly ethanol, followed by freeze-drying. In this study we therefore investigated different sampling protocols and their effects on the detected metabolite profiles in liquid chromatography-mass spectrometry (LC-MS) using an untargeted metabolomics approach. Sponges (G. barretti) were collected outside the Swedish west coast and pieces from three sponge specimens were either flash frozen in liquid nitrogen, frozen later after the collection cruise, stored in ethanol or stored in methanol. The storage solvents as well as the actual sponge pieces were analyzed, all samples were analyzed with hydrophilic interaction liquid chromatography as well as reversed phase liquid chromatography with high resolution mass spectrometry using full-scan in positive and negative ionization mode. The data were evaluated using multivariate data analysis. The highest metabolite intensities were found in the frozen samples (flash frozen and frozen after sampling cruise) as well as in the storage solvents (methanol and ethanol). Metabolites extracted from the sponge pieces that had been stored in solvent were found in very low intensity, since the majority of metabolites were extracted to the solvents to a high degree. The exception being larger peptides and some lipids. The lowest variation between replicates were found in the flash frozen samples. In conclusion, the preferred method for sampling of sponges for metabolomics was found to be immediate freezing in liquid nitrogen. However, freezing the sponge samples after some time proved to be a reliable method as well, albeit with higher variation between the replicates. The study highlights the importance of saving ethanol extracts after preservation of specimens for biology studies; these valuable extracts could be further used in studies of natural products, chemosystematics or metabolomics.

Improved Sensitivity in Hydrophilic Interaction Liquid Chromatography-Electrospray-Mass Spectrometry after Removal of Sodium and Potassium Ions from Biological Samples.

Inorganic ions, such as sodium and potassium, are present in all biological matrices and are sometimes also added during sample preparation. However, these inorganic ions are known to hamper electrospray ionization -mass spectrometry (ESI-MS) applications, especially in hydrophilic interaction liquid chromatography (HILIC) where they are retained and can be detected as adducts and clusters with mobile phase components or analytes. The retention of inorganic ions leads to co-elution with analytes and as a result ion-suppression, extensive adduct formation and problems with reproducibility. In the presented work, a sample preparation method using cation exchange solid phase extraction (SPE) was developed to trap Na+ and K+ ions from human blood plasma and head and neck cancer cells for the analysis of small cationic, anionic as well as neutral organic analytes. The investigated analytes were small, hydrophilic compounds typically in focus in metabolomics studies. The samples were analyzed using full-scan HILIC-ESI-quadrupole time of flight (QTOF)-MS with an untargeted, screening approach. Method performance was evaluated using multivariate data analysis as well as relative quantifications, spiking of standards to evaluate linearity of response and post-column infusion to study ion-suppression. In blood plasma, the reduction of sodium and potassium ion concentration resulted in improved sensitivity increased signal intensity for 19 out of 28 investigated analytes, improved linearity of response, reduced ion-suppression and reduced cluster formation as well as adduct formation. Thus, the presented method has significant potential to improve data quality in metabolomics studies.

Investigation of Equine In Vivo and In Vitro Derived Metabolites of the Selective Androgen Receptor Modulator (SARM) ACP-105 for Improved Doping Control.

Selective Androgen Receptor Modulators (SARMs) have anabolic properties but less adverse effects than anabolic androgenic steroids. They are prohibited in both equine and human sports and there have been several cases of SARMs findings reported over the last few years. The aim of this study was to investigate the metabolite profile of the SARM ACP-105 (2-chloro-4-[(3-endo)-3-hydroxy-3-methyl-8-azabicyclo[3.2.1]oct-8-yl]-3-methylbenzonitrile) in order to find analytical targets for doping control. Oral administration of ACP-105 was performed in horses, where blood and urine samples were collected over a time period of 96 h. The in vivo samples were compared with five in vitro incubation models encompassing Cunninghamella elegans, microsomes and S9 fractions of both human and equine origin. The analyses were performed using ultra-high performance liquid chromatography coupled to high resolution Q ExactiveTM OrbitrapTM mass spectrometry (UHPLC-HRMS). A total of 21 metabolites were tentatively identified from the in vivo experiments, of which several novel glucuronides were detected in plasma and urine. In hydrolyzed urine, hydroxylated metabolites dominated. The in vitro models yielded several biotransformation products, including a number of monohydroxylated metabolites matching the in vivo results. The suggested analytical target for equine doping control in plasma is a dihydroxylated metabolite with a net loss of two hydrogens. In urine, the suggested targets are two monohydroxylated metabolites after hydrolysis with ß-glucuronidase, selected both due to prolongation of the detection time and the availability of reference material from the in vitro models.

Removal of nonylphenol polyethoxylates by adsorption on polyurethane foam and biodegradation using immobilized Trametes versicolor.

Nonylphenol polyethoxylates (NPEOs) are banned in EU due to their endocrine disrupting properties. In a proof of concept study including continuous reactor lab-scale experiments, polyurethane foam (PUF)-immobilized Trametes versicolor was used to reduce the concentration levels of these compounds in an acidic nutrient solution over an 18-day period. Biodegradation and adsorption were identified as the major removal principles. A 90% removal was achieved by solely biodegradation in an experimental setup in which steady state conditions occurred, including NPEO-saturated glass and PUF surfaces. Biotransformation products containing mono- and di-ethoxylated nonylphenol, nonylphenol (NP1EO, NP2EO, NP) and nonylphenol polyethoxy carboxylates (NPECs) were tentatively identified. The maximum static NPEO adsorption capacity of PUF (determined with Erlenmeyer flask experiment) was calculated to 106 mg g-1, and the adsorption was described by the Langmuir isotherm equation. The corresponding maximum dynamic adsorption capacity (determined by continuous reactor experiment) was 100 mg g-1. These findings show that PUF is an excellent adsorbent to NPEOs. Therefore, PUF can either be used as a stand-alone adsorbent to NPEOs or as an immobilizing agent for Trametes versicolor through which a highly efficient biodegradation of these potentially harmful compounds can be achieved. The findings can be of importance in the search for alternative methods to remove NPEOs in process effluents.

Anabolic androgenic steroids exert a selective remodeling of the plasma lipidome that mirrors the decrease of the de novo lipogenesis in the liver.

INTRODUCTION: The abuse of anabolic androgenic steroids (AASs) is a source of public concern because of their adverse effects. Supratherapeutic doses of AASs are known to be hepatotoxic and regulate the lipoproteins in plasma by modifying the metabolism of lipids in the liver, which is associated with metabolic diseases. However, the effect of AASs on the profile of lipids in plasma is unknown. OBJECTIVES: To describe the changes in the plasma lipidome exerted by AASs and to discuss these changes in the light of previous research about AASs and de novo lipogenesis in the liver. METHODS: We treated male Wistar rats with supratherapeutic doses of nandrolone decanoate and testosterone undecanoate. Subsequently, we isolated the blood plasma and performed lipidomics analysis by liquid chromatography-high resolution mass spectrometry. RESULTS: Lipid profiling revealed a decrease of sphingolipids and glycerolipids with palmitic, palmitoleic, stearic, and oleic acids. In addition, lipid profiling revealed an increase in free fatty acids and glycerophospholipids with odd-numbered chain fatty acids and/or arachidonic acid. CONCLUSION: The lipid profile presented herein reports the imprint of AASs on the plasma lipidome, which mirrors the downregulation of de novo lipogenesis in the liver. In a broader perspective, this profile will help to understand the influence of androgens on the lipid metabolism in future studies of diseases with dysregulated lipogenesis (e.g. type 2 diabetes, fatty liver disease, and hepatocellular carcinoma).

An LCMS-based untargeted metabolomics protocol for cochlear perilymph: highlighting metabolic effects of hydrogen gas on the inner ear of noise exposed Guinea pigs.

INTRODUCTION: Noise-induced hearing loss (NIHL) is an increasing problem in society and accounts for a third of all cases of acquired hearing loss. NIHL is caused by formation of reactive oxygen species (ROS) in the cochlea causing oxidative stress. Hydrogen gas (H2) can alleviate the damage caused by oxidative stress and can be easily administered through inhalation. OBJECTIVES: To present a protocol for untargeted metabolomics of guinea pig perilymph and investigate the effect of H2 administration on the perilymph metabolome of noise exposed guinea pigs. METHODS: The left ear of guinea pigs were exposed to hazardous impulse noise only (Noise, n = 10), noise and H2 (Noise + H2, n = 10), only H2 (H2, n = 4), or untreated (Control, n = 2). Scala tympani perilymph was sampled from the cochlea of both ears. The polar component of the perilymph metabolome was analyzed using a HILIC-UHPLC-Q-TOF-MS-based untargeted metabolomics protocol. Multivariate data analysis (MVDA) was performed separately for the exposed- and unexposed ear. RESULTS: MVDA allowed separation of groups Noise and Noise + H2 in both the exposed and unexposed ear and yielded 15 metabolites with differentiating relative abundances. Seven were found in both exposed and unexposed ear data and included two osmoprotectants. Eight metabolites were unique to the unexposed ear and included a number of short-chain acylcarnitines. CONCLUSIONS: A HILIC-UHPLC-Q-TOF-MS-based protocol for untargeted metabolomics of perilymph is presented and shown to be fit-for-purpose. We found a clear difference in the perilymph metabolome of noise exposed guinea pigs with and without H2 treatment.

Acidic transformation of nordiazepam can affect recovery estimate during trace analysis of diazepam and nordiazepam in environmental water samples by liquid chromatography-tandem mass spectrometry.

In this study, a special interest was focused on the stability of diazepam and nordiazepam in aqueous samples at acidic and neutral pH. The aim of the study was to isolate and illustrate one of the many possible sources of error that can be encountered when developing and validating analytical methods. This can be of particular importance when developing multi-analyte methods where there is limited time to scrutinize the behavior of each analyte. A method was developed for the analysis of the benzodiazepines diazepam and nordiazepam in treated wastewater. The samples were extracted by solid phase extraction, using SPEC C18AR cartridges, and analyzed by the use of liquid chromatography, with a C18 stationary phase, coupled to tandem mass spectrometry. Environmental water samples are often acidified during storage to reduce the microbial degradation of the target compounds and to preserve the sample. In some cases, the samples are acidified before extraction. In this study, it was found that a chemical equilibrium between nordiazepam and a transformation product could cause inaccurately high extraction recovery values when the samples were stored at low sample pH. The stability of nordiazepam was shown to be low at pH 3. Within 12 days, 20% of the initial concentration of nordiazepam was transformed. Interestingly, the transformed nordiazepam was shown to be regenerated and reformed to nordiazepam during sample handling. At a sample pH of 7, diazepam and nordiazepam were stable for 12 days. It was concluded that great care must be taken when acidifying water samples containing nordiazepam during storage or extraction. The storage and the extraction should be conducted at neutral pH if no internal standard is used to compensate for degradation and conversion of nordiazepam. The developed method was validated in treated wastewater and applied for the quantification of diazepam and nordiazepam in treated wastewater samples.

Adduct formation in electrospray ionisation-mass spectrometry with hydrophilic interaction liquid chromatography is strongly affected by the inorganic ion concentration of the samples.

Hydrophilic interaction liquid chromatography (HILIC)/ electrospray ionisation-mass spectrometry (ESI-MS) has gained interest for the analysis of polar analytes in bioanalytical applications in recent years. However, ESI-MS is prone to adduct formation of analytes. In contrast to reversed phase chromatography, small inorganic ions have retention in HILIC, i.e. analytes and inorganic ions may co-elute, which could influence the adduct formation. In the present paper, it was demonstrated that the co-elution of sodium ions or potassium ions and analytes in HILIC/ESI-MS affect the adduct formation and that different concentrations of sodium ions and potassium ions in biological samples could have an impact on the quantitative response of the respective adducts as well as the quantitative response of the protonated adduct. The co-elution also lead to cluster formation of analytes and sodium formate or potassium formate, causing extremely complicated spectra. In analytical applications using HILIC/ESI-MS where internal standards are rarely used or not properly matched, great care needs to be taken to ensure minimal variation of inorganic ion concentration between samples. Moreover, the use of alkali metal ion adducts as quantitative target ions in relative quantitative applications should be made with caution if proper internal standards are not used.

Common Fatty Markers in Diseases with Dysregulated Lipogenesis.

Recent studies have reported the upregulation of a subgroup of triacylglycerides as markers of different diseases with dysregulated lipogenesis, which means that these markers are not selective. This observation has a deep impact on their use as diagnostic tools in clinical practice (e.g., markers of risk of type 2 diabetes).

Removal of diclofenac from a non-sterile aqueous system using Trametes versicolor with an emphasis on adsorption and biodegradation mechanisms.

This paper describes the search for procedures through which the xenobiotic pollutant diclofenac can be removed from non-sterile aquatic systems. Specifically, adsorption to solid supports (carriers) in combination with biodegradation by non-immobilized and immobilized white rot fungus Trametes versicolor were investigated. Batch experiments using polyurethane foam (PUF)-carriers resulted in 99.9% diclofenac removal after 4 h, with monolayer adsorption of diclofenac to carrier and glass surfaces accounting for most of the diclofenac decrease. Enzymatic reactions contributed less, accounting for approximately < 0.5% of this decrease. In bioreactor experiments using PUF-carriers, an initial 100% removal was achieved with biodegradation contributing approximately 7%. In batch experiments that utilized polyethylene-carriers with negligible immobilization of Trametes versicolor, a 98% total diclofenac removal was achieved after one week, with a biodegradation contribution of approximately 14%. Five novel enzyme-catalyzed biodegradation products were tentatively identified in the batch-wise and bioreactor experiments using full scan ultra-high-performance liquid chromatography-quadrupole/time of flight mass spectrometry. Both reduction and oxidation products were found, with the contents estimated to be at µg L-1 concentration levels.

Method selectivity evaluation using the co-feature ratio in LC/MS metabolomics: Comparison of HILIC stationary phase performance for the analysis of plasma, urine and cell extracts.

Evaluation of the chromatographic separation in metabolomics studies has primarily been done using preselected sets of standards or by counting the number of detected features. An alternative approach is to calculate each feature's co-feature ratio, which is a combined selectivity measurement for the separation (i.e. extent of co-elution) and the MS-signal (i.e. adduct formation and in-source fragmentation). The aim of this study was to demonstrate how the selectivity of different HILIC stationary phases can be evaluated using the co-feature ratio approach. The study was based on three sample types; plasma, urine and cell extracts. Samples were analyzed on an UHPLC-ESI-Q-ToF system using an amide, a bare silica and a sulfobetaine stationary phase. For each feature, a co-feature ratio was calculated and used for multivariate analysis of the selectivity differences between the three stationary phases. Unsupervised PCA models indicated that the co-feature ratios were highly dependent on type of stationary phase. For several metabolites a 15-30 fold difference in the co-feature ratio were observed between the stationary phases. Observed selectivity differences related primarily to the retention patterns of unwanted matrix components such as inorganic salts (detected as salt clusters), glycerophospholipids, and polyethylene glycols. These matrix components affected the signal intensity of co-eluting metabolites by interfering with the ionization efficiency and/or their adduct formation. Furthermore, the retention pattern of these matrix components had huge influence on the number of detected features. The co-feature ratio approach has successfully been applied for evaluation of the selectivity performance of three HILIC stationary phases. The co-feature ratio could therefore be used in metabolomics for developing selective methods fit for their purpose, thereby avoiding generic analytical approaches, which are often biased, as type and amount of interfering matrix components are metabolome dependent.

Major signal suppression from metal ion clusters in SFC/ESI-MS - Cause and effects.

The widening application area of SFC-MS with polar analytes and water-containing samples facilitates the use of quick and simple sample preparation techniques such as "dilute and shoot" and protein precipitation. This has also introduced new polar interfering components such as alkali metal ions naturally abundant in e.g. blood plasma and urine, which have shown to be retained using screening conditions in SFC/ESI-TOF-MS and causing areas of major ion suppression. Analytes co-eluting with these clusters will have a decreased signal intensity, which might have a major effect on both quantification and identification. When investigating the composition of the alkali metal clusters using accurate mass and isotopic pattern, it could be concluded that they were previously not described in the literature. Using NaCl and KCl standards and different chromatographic conditions, varying e.g. column and modifier, the clusters proved to be formed from the alkali metal ions in combination with the alcohol modifier and make-up solvent. Their compositions were [(XOCH3)n + X]+, [(XOH)n + X]+, [(X2CO3)n + X]+ and [(XOOCOCH3)n + X]+ for X = Na+ or K+ in ESI+. In ESI-, the clusters depended more on modifier, with [(XCl)n + Cl]- and [(XOCH3)n + OCH3]- mainly formed in pure methanol and [(XOOCH)n + OOCH]- when 20 mM NH4Fa was added. To prevent the formation of the clusters by avoiding methanol as modifier might be difficult, as this is a widely used modifier providing good solubility when analyzing polar compounds in SFC. A sample preparation with e.g. LLE would remove the alkali ions, however also introducing a time consuming and discriminating step into the method. Since the alkali metal ions were retained and affected by chromatographic adjustments as e.g. mobile phase modifications, a way to avoid them could therefore be chromatographic tuning, when analyzing samples containing them.

The differences in matrix effect between supercritical fluid chromatography and reversed phase liquid chromatography coupled to ESI/MS.

For many sample matrices, matrix effects are a troublesome phenomenon using the electrospray ionization source. The increasing use of supercritical fluid chromatography with CO2 in combination with the electrospray ionization source for MS detection is therefore raising questions: is the matrix effect behaving differently using SFC in comparison with reversed phase LC? This was investigated using urine, plasma, influent- and effluent-wastewater as sample matrices. The matrix effect was evaluated using the post-extraction addition method and through post-column infusions. Matrix effect profiles generated from the post-column infusions in combination with time of flight-MS detection provided the most valuable information for the study. The combination of both qualitative and semi-quantitative information with the ability to use HRMS-data for identifying interfering compounds from the same experiment was very useful, and has to the authors' knowledge not been used this way before. The results showed that both LC and SFC are affected by matrix effects, however differently depending on sample matrix. Generally, both suppressions and enhancements were seen, with a higher amount of enhancements for LC, where 65% of all compounds and all sample matrices were enhanced, compared to only 7% for SFC. Several interferences were tentatively identified, with phospholipids, creatinine, and metal ion clusters as examples of important interferences, with different impact depending on chromatographic technique. SFC needs a different strategy for limiting matrix interferences, owing to its almost reverse retention order compared to RPLC.