The functional roles of S-adenosyl-methionine and S-adenosyl-homocysteine and their involvement in trisomy 21.

The one-carbon metabolism pathway is involved in critical human cellular functions such as cell proliferation, mitochondrial respiration, and epigenetic regulation. In the homocysteine-methionine cycle S-adenosyl-methionine (SAM) and S-adenosyl-homocysteine (SAH) are synthetized, and their levels are finely regulated to ensure proper functioning of key enzymes which control cellular growth and differentiation. Here we review the main biological mechanisms involving SAM and SAH and the known related human diseases. It was recently demonstrated that SAM and SAH levels are altered in plasma of subjects with trisomy 21 (T21) but how this metabolic dysregulation influences the clinical manifestation of T21 phenotype has not been previously described. This review aims at providing an overview of the biological mechanisms which are altered in response to changes in the levels of SAM and SAH observed in DS.

First clinical evidence that trimethylsulfonium can serve as a biomarker for the production of the signaling molecule hydrogen sulfide.

BACKGROUND: Hydrogen sulfide (H2S) is established as the third gaseous signaling molecule and is known to be overproduced in down syndrome (DS) due to the extra copy of the CBS gene on chromosome 21, which has been suggested to contribute to the clinical manifestation of this condition. We recently discovered trimethylsulfonium (TMS) in human urine and highlighted its potential as a selective methylation metabolite of endogenously produced H2S, but the clinical utility of this novel metabolite has not been previously investigated. We hypothesize that the elevation of H2S production in DS would be reflected by an elevation in the methylation product TMS. METHODS: To test this hypothesis, a case-control study was performed and the urinary levels of TMS were found to be higher in the DS group (geo. mean 4.5 nM, 95 % CI 2.4-3.9) than in the control (N) group (3.1 nM, 3.5-6.0), p-value 0.01, whereas the commonly used biomarker of hydrogen sulfide, thiosulfate, failed to reflect this alteration in H2S production (15 µM (N) vs. 13 µM (DS), p-value 0.24. RESULTS: The observed association is in line with the proposed hypothesis and provides first clinical evidence of the utility of TMS as a novel and more sensitive biomarker for the endogenous production of the third gaseous signaling molecule than the conventionally used biomarker thiosulfate, which is heavily dependent on bacterial hydrogen sulfide production. CONCLUSION: This work shows that TMS must be explored in clinical conditions where altered metabolism of hydrogen sulfide is implicated.

A time-course investigation of the human urinary excretion of the hydrogen sulfide biomarker trimethylsulfonium.

Hydrogen sulfide is a toxic gas but also recognized as an endogenously produced metabolite in humans playing key roles. We previously identified trimethylsulfonium, which can be a methylation product of hydrogen sulfide but the stability in the production of trimethylsulfonium has not been investigated. In the present work, the intra- and inter-individual variability in the excretion of trimethylsulfonium over 2 months in a group of healthy volunteers was investigated. Urinary levels of trimethylsulfonium (mean: 56 nM, 95% CI: 48-68 nM) were > 100-fold lower than the conventional hydrogen sulfide biomarker thiosulfate (13 µM, 12-15 µM) and the precursor for endogenous hydrogen sulfide production cystine (47 µM, 44-50 µM). There was no correlation between urinary trimethylsulfonium and thiosulfate. Higher intra-individual variability in the excretion of trimethylsulfonium (generally 2-8 fold) than that for cystine (generally 2-3 fold) was found. Trimethylsulfonium displayed significant inter-individual variability with two concentration clusters at 117 nM (97-141) and 27 nM (22-34). In conclusion, the observed inter- and intra-individual variability must be considered when using urinary trimethylsulfonium as a biomarker.

Determination of Phosphoethanolamine in Urine with HPLC-ICPMS/MS Using 1,2-Hexanediol as a Chromatographic Eluent.

The importance of element-selective detection with inductively coupled plasma mass spectrometry (ICPMS) has been significantly increased in recent years following the introduction of tandem ICPMS (ICPMS/MS), which unlocked access to nonmetal speciation analysis. However, nonmetals are ubiquitous, and the feasibility of nonmetal speciation analysis in matrices with complex metabolomes is yet to be demonstrated. Herein, we report the first phosphorous speciation study by HPLC-ICPMS/MS in a human sample, namely, urine, involving the determination of the natural metabolite and biomarker phosphoethanolamine. A simple one-step derivatization procedure was employed to enable the separation of the target compound from the hydrophilic phosphorous metabolome in urine. The challenge of eluting the hydrophobic derivative under ICPMS-compatible chromatographic conditions was addressed by employing hexanediol, a novel chromatographic eluent recently described in our previous work but has not yet been exploited in a real-world application. The developed method features fast chromatographic separation (<5 min), no need for an isotopically labeled internal standard, and an instrumental LOD of 0.5 µg P L-1. The method was evaluated for recovery (90-110%), repeatability (RSD ±5%), and linearity (r2 = 0.9998). The method accuracy was thoroughly examined by comparing with an independently developed method based on HPLC-ESIMS/MS without derivatization, where agreement was found within ±5-20%. An application is presented to gain first insight into the variability in the human excretion of phosphoethanolamine, which is key for the interpretation of its levels as a biomarker, by repeated urine collection from a group of volunteers over 4 weeks.

Trace determination of the hydrogen sulfide biomarker thiosulfate in human urine by HPLC coupled with element selective ICPMS/MS detection.

Hydrogen sulfide is a toxic gas but also established as a naturally occurring gaseous signaling molecule in humans, playing key physiological roles with particular involvement in lung disease including COVID-19. Thiosulfate is the conventional biomarker of hydrogen sulfide and is excreted in human urine at low micromolar levels. Thiosulfate is amenable to detection by the element-selective inductively coupled plasma tandem mass spectrometry (ICPMS/MS), but sulfur speciation in human samples at trace levels is challenging due to the high complexity of human sulfur metabolome and the utility of this detector under such settings has not been demonstrated. We report a method for thiosulfate determination in human urine at trace physiological levels by HPLC-ICPMS/MS. The method involved one-step derivatization to improve chromatographic behavior followed by direct injection. The instrumental limit of detection was 1.4 µg S L-1 (0.02 µM or 0.1 pmol). In a group of samples from volunteers (n = 24), measured thiosulfate concentrations in the diluted urine matrix were down to 8.0 µg S L-1 with a signal-to-noise ratio >10. The method was validated for recovery (80-110%), repeatability (RSD% <5%), and linearity (r2 = 0.9999, at a tested working concentration range of 0.01-1.0 mg S L-1), and the accuracy was assessed by comparing with HPLC-ESIMS/MS which showed agreement within ±20%. This work demonstrates the applicability of HPLC-ICPMS/MS for sulfur speciation at trace levels in a matrix with complex sulfur metabolome as human urine and provides a sensitive method for the determination of the hydrogen sulfide biomarker.

Trimethylselenonium ion determination in human urine by high-performance liquid chromatography-hydride generation-atomic fluorescence spectrometry optimization of the hydride generation step.

This work describes the intricacies of the determination of the trimethylselenonium ion (TMSe) in human urine via high-performance liquid chromatography-hydride generation-atomic fluorescence spectrometry (HPLC-HG-AFS). By definition, this technique requires that the separated TMSe can be online converted into a volatile compound. Literature data for the determination of TMSe via the hydride generation technique are contradictory; i.e., some authors claim that direct formation of volatile compounds is possible under reduction with NaBH4, whereas others reported that a digestion step is mandatory prior to conversion. We studied and optimized the conditions for online conversion by varying the mobile phase composition (pyridine, phosphate, and acetate), testing different reaction coils, and optimizing the hydride generation conditions, although technically no hydride (H2Se) is formed but a dimethylselenide (DMSe). The optimized conditions were used for the analysis of 64 urine samples of 16 (unexposed) volunteers and the determination of low amounts of TMSe (LOD = 0.2 ng mL-1). Total (specific gravity-corrected) selenium concentrations in the urine samples ranged from 7.9 ± 0.7 to 29.7 ± 5.0 ng mL-1 for individual volunteers. Four volunteers were characterized as TMSe producers (hINMT genotype GA) and 12 were non-producers (hINMT genotype GG). Urine of TMSe producers contained 2.5 ± 1.7 ng mL-1 of TMSe, compared to 0.2 ± 0.2 ng mL-1 for non-producers.

Variability in Background Urinary Concentrations of the Hydrogen Sulfide Biomarker Thiosulfate.

Hydrogen sulfide is a toxic gas at high concentrations but has recently attracted attention as a naturally produced gaseous signaling molecule in various tissues of the human body, playing key physiological roles at low nanomolar concentrations. This has wide implications for chronic exposure to this gas in air at low levels far below toxicity. Thiosulfate is the currently used biomarker for exposure to hydrogen sulfide via inhalation but has been mainly employed for acute exposure. It is unknown how background thiosulfate concentrations vary on an intraindividual and interindividual basis in humans at normal ambient hydrogen sulfide levels (<1 µg m-3), which is key for the interpretation of its levels as biomarker for low-level hydrogen sulfide exposure. In the current work, the variability in thiosulfate urinary excretion in a total of 168 urine samples collected from eight volunteers over a period of 8 weeks was investigated. The determination of thiosulfate in urine was carried out by UHPLC-MS/MS. The total average concentration ± SD was 16 ± 6 µM (n = 168). Average urinary thiosulfate concentrations in the studied volunteers were within the range of 10-20 µM, but it was found that urinary thiosulfate can show significant day-to-day and week-to-week variability in some individuals (up to 10-fold), despite adjusting for urine specific gravity. In light of the presented variability data and previous studies about the lack of consistent response of thiosulfate to low levels of hydrogen sulfide inhalation exposure, and based on a review of the biochemistry of the production of thiosulfate and its various biological sources, it can be argued that thiosulfate might not be suitable as a biomarker for chronic environmental exposure to low levels of hydrogen sulfide via inhalation.

Elution with 1,2-Hexanediol Enables Coupling of ICPMS with Reversed-Pase Liquid Chromatography under Standard Conditions.

The inductively coupled plasma mass spectrometry (ICPMS) has been attracting increasing attention for many applications as an element-selective chromatographic detector. A major and fundamental limitation in coupling ICPMS with liquid chromatography is the limited compatibility with organic solvents, which has so far been addressed via a tedious approach, collectively referred to as the "organic ICPMS mode", that can decrease detection sensitivity by up to 100-fold. Herein, we report 1,2-hexanediol as a new eluent in high-performance liquid chromatography-ICPMS which enables avoiding the current limitations. Unlike commonly used eluents, 1,2-hexanediol was remarkably compatible with ICPMS detection at high flow rates of 1.5 mL min-1 and concentrations of at least 30% v/v, respectively, under the standard conditions and instrumental setup normally used with 100% aqueous media. Sensitivity for all tested elements (P, S, Cl, Br, Se, and As) was enhanced with 10% v/v 1,2-hexanediol relative to that of 100% aqueous media by 1.5-7-fold depending on the element. Concentrations of 1,2-hexanediol at ≤30% v/v were superior in elution strength to concentrations at >90% v/v of the common organic phases, which greatly decreases the amount of carbon required to elute highly hydrophobic compounds such as lipids and steroids, enabling detection at ultra-trace levels. The proposed approach was applied to detect arsenic-containing fatty acids in spiked human urine, and detection limits of <0.01 µg As L-1 were achieved, which is >100-fold lower than those previously reported using the organic ICPMS mode. Nontargeted speciation analysis in Allium sativum revealed the presence of a large number of hydrophobic sulfur-containing metabolomic features at trace levels.

Rapid determination of hydrogen sulfide-related metabolites in human urine by UHPLC-ESI-MS/MS with fluorinated ion-pairing reagents.

Although primarily considered as a toxic gas, hydrogen sulfide is naturally produced in humans from the amino acid cysteine in low quantities and is commonly referred to as the "third gaseous signaling molecule". There is a need to establish reliable biomarkers for exposure to hydrogen sulfide in human matrices not only at toxic levels but also at trace levels. Herein, we report optimized analytical methods for the direct determination of three compounds related to hydrogen sulfide metabolism in human urine without sample preparation; namely, trimethylsulfonium, thiosulfate, and cystine. The methods were based on ion-pair ultra-high performance liquid chromatography (UHPLC) with an anionic fluorinated acid or a cationic fluoroalkylamine, a member of a new generation of ion-pairing reagents, and detection by the electrospray ionization tandem mass spectrometry (ESI-MS/MS). Rapid chromatographic separation was achieved in 2-3 min with limits of detection of 0.1, 2.5, and 50 nmol L-1 (nM) for trimethylsulfonium, cystine, and thiosulfate, respectively. Isotopically labeled internal standards were used for each analyte to account for matrix effects and qualifier ions were employed to ensure selectivity. The method was validated for recovery (generally within 80-120%) and repeatability (RSD% 1.0-10%), and applied to investigate the normal concentrations of the three analytes in forty morning first-pass urine samples of healthy volunteers.

The determination of the Paraxanthine/Caffeine ratio as a metabolic biomarker for CYP1A2 activity in various human matrices by UHPLC-ESIMS/MS.

The Cytochrome P450 CYP1A2 is a central enzyme in the metabolism of drugs and xenobiotics. The overall activity of this enzyme is influenced by a complex array of biochemical, dietary, and genetic factors. One of the simplest ways to probe the overall output of CYP1A2 is to measure the ratio between the concentration of a precursor and a product of its activity. With the growing interest in the Paraxanthine/Caffeine ratio, the need arises to develop improved analytical methods specifically optimized for the rapid and sensitive determination of paraxanthine and caffeine in biological samples. We report a new optimized method for the determination of caffeine and paraxanthine in various human matrices. The method involved direct determination following protein precipitation based on ultra high performance liquid chromatographic separation with tandem mass spectrometric detection (UHPLC-ESIMS/MS). The method offers an improvement in the detection limit over previously published methods by at least 10-fold (0.1 pg), rapid chromatographic separation (ca. 5 min), the utilization of a green chromatographic solvent (5% v/v ethanol), direct determination with little sample preparation, and the employment of isotopically labeled internal standards and qualifier ions to ensure accuracy. Method validation in urine, saliva, and plasma was performed by spiking at various concentration levels where the recovery and repeatability were within ±15% and ±10%, respectively. The method was applied to investigate the levels of caffeine and paraxanthine in volunteers following controlled caffeine administration and to investigate the inter- and intra-individual variability in the paraxanthine/caffeine ratio in volunteers following an unrestricted caffeine diet. In conclusion, the developed UHPLC-ESIMS/MS method is optimized specifically for the simultaneous determination of the paraxanthine/caffeine ratio in multiple biological matrices, offers several advantages over the current methods, and is well suitable for application in large clinical studies.

Parallel and comparative non-targeted metabolomic speciation analysis of metalloids and their non-metal analogues by HPLC-ICPMS/MS in mushrooms.

With the introduction of tandem mass spectrometry to inductively coupled plasma mass spectrometry (ICPMS/MS), the potential for non-targeted elemental metabolomic analysis has been expanded to many non-metals of pivotal biological importance. Arsenic and selenium are trace elements that share chemical similarity with the non-metals phosphorus and sulfur, respectively, and this similarity can be exploited to gain more insight into the incompletely understood biological significance of these metalloids and the evolution of their biochemical pathways. As a proof of concept, we show the applicability of HPLC-ICPMS/MS for non-targeted and parallel speciation analysis of arsenic, selenium, phosphorus, and sulfur in mushrooms-metabolically diverse organisms. Incredibly contrasting levels of diversity were found in the metabolomic profiles of the four investigated elements among the various species along with sharp discrepancies among related elements (e.g. phosphorous vs. arsenic) in certain mushroom species. The present work shows that ICPMS/MS offers a new dimension in non-targeted metabolomic analysis and enables a unique comparative approach in investigating and tracking the biochemistry of related elements in moderately complex organisms.

Is the water disinfection by-product dichloroacetic acid biosynthesized in the edible mushroom Russula nigricans?

We report the first halogen speciation analysis study by high performance liquid chromatography coupled with inductively coupled plasma tandem mass spectrometry (HPLC-ICPMS/MS) in the fruiting bodies of various mushroom species. Non-targeted speciation analysis revealed the occurrence of dichloroacetic acid (DCAA) in the edible mushroom Russula nigricans. Multiple samples of this mushroom (n = 5) collected from different geographic non-industrial regions in two different countries confirmed the consistent presence of this species at a relatively narrow concentration range (23-37 mg kg-1), whereas no other chlorinated acetic acid (e.g. chloroacetic acid and trichloroacetic acid) was detected. Neither DCAA nor any other chlorinated acetic acid were detected in any of the other mushroom species investigated in the present study, including seven different mushroom species of the same genus Russula, even though all mushrooms were collected from the same non-industrial geographic regions. Together with the previously reported biological activities of DCAA, these findings collectively suggest biosynthesis of this compound as an explanation for its dominant presence in R. nigricans, and constitute the first example of the dominant natural occurrence of this compound over other chlorinated acetic acids in a living organism. This may warrant a change in our view of the occurrence of dichloroacetic acid in nature, where primarily considered as a pollutant arising from water disinfection.

Fluorinated carboxylic acids as "ion repelling agents" in reversed-phase chromatography.

Fluorinated carboxylic acids have been in use as ion-pairing reagents for over three decades. It has been observed that ion-pairing reagents not only increase the retention of oppositely charged analytes on reversed-phase HPLC columns but also decrease the retention of similarly charged analytes; these latter effects, however, have not been thoroughly investigated for the fluorinated carboxylic acids, and the application of these reagents has been rather restricted to their ion-pairing capacity to separate basic analytes. In the present study, we report a systematic investigation about the effects of three fluorinated carboxylic acids (trifluoroacetic acid (TFA), pentafluoropropionic acid (PFPA), and heptafluorobutyric acid (HFBA)) on the retention and selectivity of the separation of halogenated carboxylic acids and sulfonic acids by reversed-phase chromatography with an inductively coupled plasma mass spectrometry detector (ICPMS). Several eluents were tested and compared at different concentrations (0-100 mM) and pH values, including sulfate, nitrate, phosphate, oxalate, TFA, PFPA, and HFBA. The fluorinated carboxylic acids resulted in a consistent decrease in the retention factors (up to ca. 9-fold with HFBA) in a concentration dependent manner, which plateaued at around 50 mM. Significant improvement of the peak symmetry of the chromatographed acids was also observed. We highlight the advantages of incorporating the fluorinated carboxylic acids in modifying the selectivity and retention of organic acids in reversed phase chromatography in general, and particularly when employing chromatographic detectors with limited compatibility with organic mobile phases such as the ICPMS.

Fluoroalkylamines: Novel, Highly Volatile, Fast-Equilibrating, and Electrospray Ionization-Mass Spectrometry Signal-Enhancing Cationic Ion-Interaction Reagents.

A new class of cationic ion-interaction reagents for reversed-phase chromatography is introduced in the present work. Compounds belonging to a homologous series of linear fluoroalkyl chains including trifluoroethylamine (TFEAm), pentafluoropropylamine (PFPAm), heptafluorobutylamine (HFBAm), and nonafluoropentylamine (NFPAm) were tested and compared with ammonia and triethylamine (TEA) for the separation of selected organic acids of general interest such as the herbicides glyphosate, ethephon, and fosamine and arsenic metabolites methylarsonic acid and dimethylarsinic acid as well as other compounds. Depending on the carbon and fluorine atom number, the fluoroalkylamines were shown to be effective cationic ion-interaction reagents, significantly enhancing the retention of organic acids on a C18 reversed-phase column. Contrary to the general behavior of ion-interaction reagents (a broader term than ion-pairing reagent), significant (up to 5-fold) and consistent enhancement in the electrospray ionization mass spectrometry signal (ESI-MS) was observed relative to ammonia and triethylamine. Overall, among the tested series HFBAm was found to offer the best overall properties among the tested series as it provided a good compromise between column equilibration time (ca. 25 column volumes) and retention behavior (up to a 10-fold increase in the retention factor of acids relative to ammonia) while providing the same general advantages found for the fluoroalkylamines such as fast washout times from the ESIMS system (ca. 30 min) and a 3-5-fold signal enhancement. The fluoroalkylamines are a new class of cationic ion-interaction reagents with clear advantages over the currently employed alkylamines and may revive the general interest in ion-interaction chromatography.

Simultaneous Determination of Chlorinated and Brominated Acetic Acids in Various Environmental Water Matrixes by High-Performance Liquid Chromatography-Inductively Coupled Plasma Tandem Mass Spectrometry without Sample Preparation.

The halogenated acetic acids (HAAs) are generally considered as environmental contaminants and are suspected to pose a major public health concern. The inductively coupled plasma mass spectrometry (ICPMS) has been improved by coupling with the tandem mass spectrometry technology (ICPMS/MS), enabling ultratrace determination of heteroatoms. There have been few reports about the determination of chlorine-containing analytes by high-performance liquid chromatography (HPLC)-ICPMS/MS but none about utilizing this technique for the speciation analysis of organic halogenated compounds in environmental matrixes. We report a rapid method for the simultaneous determination of up to nine chlorinated and brominated acetic acids by HPLC-ICPMS/MS in Austrian surface, ground, and tap water. The chromatographic separation of the main five regulated haloacetic acids (so-called HAA5: chloroacetic acid, dichloroacetic acid, trichloroacetic acid, bromoacetic acid, and dibromoacetic acid) could be achieved in <6 min with limits of detection of 1.4-1.6 µg Cl L-1 and 0.8-1.5 µg Br L-1 for the chlorinated and brominated acetic acids, respectively. The method was validated through recovery experiments at four concentration levels (10-500 µg L-1) as well as by analyzing the U.S. Environmental Protection Agency (EPA) 552.2 CRM (certified reference material) in pure water and in three different water matrixes (tap, river, and groundwater), and thereby validated for repeatability (RSD% 1-10%), accuracy (±1.0-15%), and linearity (r2 = 0.9996-0.9999). The method fulfills the regulatory concentration limits by the EPA for HAA5 [maximum contaminant level (MCL) 60 µg L-1] and the limits currently being reviewed by the European Union for HAA9 (80 µg L-1) and demonstrates the advantages of HPLC-ICPMS/MS for the analysis of environmental water samples for halogen-tagged contaminants.

HPLC-ICPMS/MS shows a significant advantage over HPLC-ICPMS for the determination of perchlorate in ground, tap, and river water.

Perchlorate is an environmental contaminant that originates from anthropogenic as well as natural sources. The health effects of perchlorate with respect to the thyroid function are well known. The capabilities of the inductively coupled plasma mass spectrometry (ICPMS) has been recently expanded by coupling with tandem mass spectrometry. The advantages of HPLC-ICPMS/MS has not yet been thoroughly assessed for chlorine speciation analysis. We report a rapid and simple method for perchlorate determination in ground, tap, and river water and we show that the ICPMS/MS offers as a chromatographic detector at least 10-fold improvement in the detection limit relative to single quadrupole ICPMS. The achieved limit of detection was 0.3 µg Cl L-1 perchlorate and could be yet improved by simple freeze-drying (down to ca. 50 ng Cl L-1, based on the river water matrix), which is comparable with HPLC-ESIMS/MS, with the advantage of eliminating the need for the 18O-perchlorate isotopically labeled internal standard. The method was validated in the various water matrices in terms of recovery at levels 5.0-250 µg Cl L-1 (±10%), repeatability (RSD<5%), and linearity (r2 = 0.9999), and revealed the presence of trace levels in the Mur river water (0.33 ±â€¯0.04 µg Cl L-1). Carbon was found to significantly decrease signal response for chlorine, and we shed light on the general effects of carbonate on chlorine speciation in hard water analysis.

Exploring the sulfur species in wine by HPLC-ICPMS/MS.

The recent development of the inductively coupled plasma tandem mass spectrometry (ICPMS/MS) offers exciting opportunities for non-metal speciation analysis. In this work, we report the first sulfur speciation study in wine by HPLC-ICPMS/MS. The major sulfur compounds were found to be sulfate (50-81 mg S L-1) followed by sulfite (18-24 mg S L-1 free sulfite & 41-63 mg S L-1 after base hydrolysis). We detected small amounts of methionine in wine (0.5-1.0 mg S L-1); Thiosulfate was below the detection limit of 0.1 mg S L-1 (injection volume 1.0 µL). A few unknown compounds (collectively 1.0-2.0 mg S L-1) were observed in the chromatograms. However, the sum of detected species accounted for only 65-77% of total sulfur concentration (105-165 mg S L-1). To investigate this gap between total sulfur and the sum of detected peaks, we performed several chemical treatments of wine including hydrolysis, oxidation, and enzymatic digestion and then re-examined the distribution of sulfur species. Furthermore, we developed a chromatographic method involving the direct determination of sulfite in white and red wine. The recovery was within the range of 98-106% and repeatability (RSD%) in the range of 6.9-9.4% in regular and "sulfite-free" red and white wine. The pH of the mobile phase drastically influenced the chromatographic behavior of the sulfite ion. At neutral pH a significant tailing and a more than 10-fold loss in peak area was observed. This effect was not previously reported and must be taken into consideration when attempting direct chromatography of sulfite.

The identification and chromatographic separation of a new highly analogous impurity of the active pharmaceutical ingredient icatibant.

Icatibant is a peptidomimetic drug serving as a bradykinin-receptor antagonist and is approved in Europe and the United States for the treatment of hereditary angioedema attacks. We have detected an impurity with a high structural similarity to icatibant in pharmaceutical dosage forms using an optimized chromatographic method based on reversed phase high performance liquid chromatography with UV detection. The abundance of the impurity was around 1% relative to the icatibant peak following storage at room temperature for 1 month, and raised up to ~16% upon temperature stressing at 100 °C. The impurity was isolated by fraction collection and further purified by solid phase extraction for structural identification. NMR and high resolution mass spectrometric analyses revealed that this impurity results from isomerization in the N-terminal single amino acid residue. The new impurity may warrant particular attention due to its exceptional similarity to the active ingredient icatibant.

Direct speciation analysis of organophosphorus environmental pollutants in water by HPLC-ICPMS/MS.

A new and optimized HPLC-ICPMS/MS method for the simultaneous determination of five of the most common phosphorous environmental pollutants in various water matrices has been developed, including aminomethylphosphonic acid (AMPA), glufosinate, glyphosate, fosamine, and ethephon. We show that the ICPMS/MS confers an average of 20-fold improvement in the detection limit (0.1-0.3 µg P L-1) relative to comparable methods based on single quadrupole ICPMS detection, without the incorporation of any sample purification or preconcentration step. The method was validated by determining the intra-day repeatability (< 6%), inter-day repeatability (< 10%) and recovery (86-112%) of 4 levels of spiking (5.0-250 µg L-1). The developed method involved utilizing the carbon enhancement effect which yielded up to 3-fold increase in sensitivity. A signal suppression effect exerted by the carbonate content of the analyzed water samples was identified, and should be generally taken into consideration when analyzing untreated hard water samples by HPLC-ICPMS. The developed method can be an important contribution in order to keep up with increasingly more stringent future regulations for the levels of the analyzed pollutants given their rising health concerns.