Ionization Liquid Chromatography Tandem Mass Research Articles

Photodegradation profiling of nitrendipine: evaluation of active pharmaceutical ingredient, tablets and its altered forms.

Nitrendipine (NTR) is a dihydropyridine drug, which is well-known as a photodegradable pharmaceutical. However, the photochemical reaction of NTR has not been evaluated in detail from now. In this study, we perform the photodegradation profiling of NTR for the elucidation of its photochemical behavior. NTR amounts during ultraviolet light (UV) irradiation were monitored using high performance liquid chromatography (HPLC). NTR was photodegraded almost completely within 24h along with the generation of some photoproducts. Structural determination of two NTR photoproducts were carried out by means of electrospray ionization liquid chromatography tandem mass spectrometry (LC-ESI-MS/MS). Obtained results from this study clarified one novel NTR photoproduct, a nitroso pyridine analogue, in addition to a pyridine analogue. Furthermore, photodegradation pathway of NTR was speculated based on chemical structures of NTR photoproducts to clarify its photochemical behavior. It was proposed that a singlet oxygen molecule might withdraw two hydrogen radicals resulting in the form of a pyridine analogue, and the following reduction of its nitro group might produce a nitroso pyridine analogue. Finally, we evaluated the photostability of NTR tablets and its altered forms, indicating that the change of the dosage form led to a decrease of the photostability of NTR tablets. The obtained results will be helpful for the additional research to evaluate the effect of NTR photodegradation on its own biological activities.

Multicenter evaluation of use of dried blood spotcompared to conventional plasma in measurements of globotriaosylsphingosine (LysoGb3) concentration in 104 Fabry patients.

Fabry disease (FD) is an X-linked lysosomal storage disorder, resulting from a deficiency of the enzyme α-galactosidase A, responsible for breaking down glycolipids such as globotriaosylceramide and its deacylated derivative, globotriaosylsphingosine (LysoGb3). Here, wecompare the levels of LysoGb3 in dried blood spots (DBS) and plasma in patients with classic and late-onset phenotypes. LysoGb3 measurements were performed in 104 FD patients, 39 males and 65 females. Venous blood was collected. A portion was spotted onto filter paper and another portion separated to obtain plasma. The LysoGb3 concentrations in DBS and plasma were determined by highly sensitive electrospray ionization liquid chromatography tandem mass spectrometry. Agreement between different matrices was assessed using linear regression and Bland Altman analysis. The method on DBS was validated by evaluating its precision, accuracy, matrix effect, recovery, and stability. The analytical performances were verified by comparison of a total of 104 paired DBS and plasma samples from as many FD patients (representing 46 GLA variants). There was a strong correlation between plasma and the corresponding DBS LysoGb3 concentrations, with few exceptions. Discrepancies were observed in anemic patients with typically low hematocrit levels compared to the normal range. The method proved to be efficient for the rapid analysis of LysoGb3. DBS provides a convenient, sensitive, and reproducible method for measuring LysoGb3 levels for diagnosis, initial phenotypic assignment, and therapeutic monitoring in patients with FD.

Solid phase extraction and LC-MS/MS quantification of ibandronate in human plasma

Purpose: To develop and validate a simple, highly sensitive and accurate method for the quantification of ibandronate (IBN) in human plasma.Methods: Electrospray ionization liquid chromatography tandem mass spectrometry (LC-MS/MS) in positive ion mode was applied to obtain optimum signals. The parent ion was acquired under collisionactivated dissociation conditions, and the abundant fragments used to design multiple reaction monitoring experiments for monitoring two ibandronate transitions (m/z 376 to m/z 114, and m/z 376 to m/z 250). The IBN was isolated from plasma with weak anion exchange solid phase extraction columns with ‘on-cartridge’ derivatization using tri-methylsilyl-diazomethane (TMSDZ) reagent to convert IBN to tetra-methyl derivative.Results: The studied drug was successfully extracted from plasma samples without any interference at a retention time of 3.2 min. The matrix effect averaged 110 %, indicating that endogenous materials had little effect on ionization. The relationship between plasma analyte concentration and IBN signal area was satisfactorily linear, with correlation coefficient (r2) ranging from 0.9817 to 0.9942 in the concentration range of 0.5 – 200 ng/ml. The lower and upper limits of quantification (LLOQ and ULOQ) for IBN were 0.5 and 200 ng/ml, respectively. Relative recovery of IBN from plasma after extraction and derivatization at 3 distinct concentrations was 83.93 to 85.06 %, relative to standard solutions. The ranges of intra- and inter-day accuracies of quantification of quality controls were 89.39 - 106.40 %, and 90.50 - 107.96 %, respectively. Processed plasma IBN extracts were stable in autosampler at 4 0C (91.12 to 103.49%). Long-term stability in plasma after 30 days at -24 0C ranged from 89.52 to 113.18 %.Conclusion: This validated LC-MS/MS method can be successfully applied for determination of IBN in pharmacokinetic studies. It is a sensitive and specific assay for plasma IBN in bioequivalence studies.
 Keywords: Ibandronate, LC-MS/MS, Validation, Derivatization, Solid-phase extraction

The role of ethanol in fatalities in Jeddah, Saudi Arabia

In Saudi Arabia, alcohol consumption is prohibited by law, but interpreting postmortem ethanol can be complicated by its postmortem production. This study developed and validated a method using headspace gas chromatography with flame ionization detection and liquid chromatography tandem mass spectroscopy to detect ethanol and its polar metabolites (ethyl glucuronide [EtG] and ethyl sulfate [EtS]) in postmortem blood and urine specimens, respectively. All calibration curves were linear with coefficients of determination greater than 0.999. The limits of detection ranged 4.5–5.0mg/dL for ethanol and 0.05–0.06mg/L for EtG and EtS. The limits of quantification were 10.0mg/dL for ethanol and 0.075mg/L for EtG and EtS. Within-run precision was less than 11% for all analytes of interest. Matrix effects for EtG and EtS ranged 3–47%. After excluding matrix effects, analytical recoveries ranged 72–100%. This validated method was then used for routine postmortem forensic toxicology analyses in 592 routine postmortem cases to distinguish between antemortem ethanol consumption and its postmortem microbial formation. Among them, 98 blood samples (17%) were positive for ethanol or its polar metabolites. Thirty-two of these cases (33%) were positive for EtG and EtS and therefore due to antemortem ethanol consumption. The remaining 66 (67%) cases were negative for both EtG and EtS and therefore due to postmortem ethanol synthesis. Because this is the first study to report the problem of alcohol consumption in Saudi Arabia, further studies are essential for validating these findings.

Blood Proteomics-Based Detection of Upregulated Lipid Metabolism and Immune Dysfunction in an Elite Adventure Athlete Trekking Across Antarctica

Abstract Objectives Proteomics when combined with psychological, nutrition, and performance measures may serve as a useful monitoring system for immune dysfunction, training distress, and exercise-induced muscle damage and exhaustion in athletes. Global proteomics monitoring of an elite adventure athlete (age 33 years) was conducted over a 28-week period that culminated in the successful, unassisted 2-month trek across Antarctica (1500 km). Methods Training distress was monitored weekly using the 19-item, validated Training Distress Scale (TDS). Weekly dried blood spot (DBS) specimens were collected via fingerprick blood drops onto standard blood spot cards. DBS proteins were measured with nano-electrospray ionization liquid chromatography tandem mass spectrometry (nanoLC-MS/MS) in data-independent acquisition (DIA) mode, and 712 proteins were identified and quantified. Results The participant experienced a decrease of 11.4 kg in body mass during the Antarctica trek. The 28-week period was divided into time segments based on TDS scores, and a contrast analysis between weeks 5–8 (low TDS) and weeks 20–23 (high TDS, last month of Antarctica trek) showed that 31 proteins (n = 20 immune related, n = 14 nutrition related with n = 8 in dual roles) were upregulated and 35 (n = 17 immune related) were downregulated. Protein-protein interaction (PPI) networks and gene ontology (GO) biological process analysis supported an increase in plasma lipoprotein particle remodeling, regulation of lipid transport, retinoid metabolic process, and vitamin transport due to high energy intake (7048 kcal/d). PPI networks also supported a dichotomous immune response. GO terms for the upregulated immune proteins showed an increase in regulation of the immune system process, especially inflammation, complement activation, and leukocyte mediated immunity. GO terms for the downregulated immune-related proteins indicated a decrease in several aspects of the overall immune system process including neutrophil degranulation and the antimicrobial humoral response. Conclusions These proteomics data support a dysfunctional immune response in an elite adventure athlete during a sustained period of mental and physical distress, high energy intake, and significant loss of body mass while trekking solo across Antarctica. Funding Sources Standard Process, Inc., Palmyra, WI.

Proteomics-Based Detection of Immune Dysfunction in an Elite Adventure Athlete Trekking Across the Antarctica.

Proteomics monitoring of an elite adventure athlete (age 33 years) was conducted over a 28-week period that culminated in the successful, solo, unassisted, and unsupported two month trek across the Antarctica (1500 km). Training distress was monitored weekly using a 19-item, validated training distress scale (TDS). Weekly dried blood spot (DBS) specimens were collected via fingerprick blood drops onto standard blood spot cards. DBS proteins were measured with nano-electrospray ionization liquid chromatography tandem mass spectrometry (nanoLC-MS/MS) in data-independent acquisition (DIA) mode, and 712 proteins were identified and quantified. The 28-week period was divided into time segments based on TDS scores, and a contrast analysis between weeks five and eight (low TDS) and between weeks 20 and 23 (high TDS, last month of Antarctica trek) showed that 31 proteins (n = 20 immune related) were upregulated and 35 (n = 17 immune related) were downregulated. Protein–protein interaction (PPI) networks supported a dichotomous immune response. Gene ontology (GO) biological process terms for the upregulated immune proteins showed an increase in regulation of the immune system process, especially inflammation, complement activation, and leukocyte mediated immunity. At the same time, GO terms for the downregulated immune-related proteins indicated a decrease in several aspects of the overall immune system process including neutrophil degranulation and the antimicrobial humoral response. These proteomics data support a dysfunctional immune response in an elite adventure athlete during a sustained period of mental and physical distress while trekking solo across the Antarctica.

Proteomic analysis and white spot syndrome virus interaction of mud crab (Scylla olivacea) revealed responsive roles of the hemocytes

White spot disease (WSD) is a highly virulent viral disease in shrimps. Clinical signs and high mortality of WSD is generally observed after a few days of infection by White Spot Syndrome virus (WSSV). Mud crabs are the major carrier and persistent host for the WSSV. However, an elucidation of viral interaction and persistent mode of WSSV infection in mud crab is still limited. We investigated the defensive role of mud crab (Scylla olivacea) hemocytes against WSSV infection by using comparative proteomic analysis coupled with electrospray ionization liquid chromatography tandem mass spectrometry (ESI-LC/MS/MS). The proteomic maps of expressed proteins obtained from WSSV infected hemocytes revealed differential proteins related to various biological functions, including immune response, anti-apoptosis, endocytosis, phosphorylation signaling, stress response, oxygen transport, molting, metabolism, and biosynthesis. Four distinctive cell types of crab hemocytes: hyaline cells (HC), small granular cells (SGC), large granular cells (LGC) and mixed granular cells (MGC) were found susceptible to WSSV. However, immunohistochemistry analysis demonstrated a complete replication of WSSV only in SGC and LGC. WSSV induced apoptosis was also observed in HC, SGC and MGC except for LGC. These results suggested that HC and MGC may undergo apoptosis prior to a complete assembly of virion, while SGC is more susceptible showing higher amplification and releasing of virion. In contrast, WSSV may inhibit apoptosis in infected LGC to stay in latency. This present finding provides an insight for the responsive roles of crustacean hemocyte cells involved in molecular interaction and defense mechanism against WSSV.

Adsorption and photocatalytic oxidation of ibuprofen using nanocomposites of TiO2 nanofibers combined with BN nanosheets: Degradation products and mechanisms

This study investigated the adsorption and photocatalytic activity of titanium dioxide (TiO2)-boron nitride (BN) nanocomposites for the removal of contaminants of emerging concern in water using ibuprofen as a model compound. TiO2 nanofibers wrapped by BN nanosheets were synthesized by electrospinning method. Characterization of the nanocomposite photocatalysts indicated that the BN nanosheets improved the light absorbance and reduced the recombination of the photoexcited charge carriers (e− and h+). The photocatalytic oxidation products and mechanisms of ibuprofen by the TiO2-BN catalysts were elucidated using a multiple analysis approach by high performance liquid chromatography, ultraviolet absorbance, dissolved organic carbon, fluorescence excitation-emission matrices, and electrospray ionization–liquid chromatography–tandem mass spectrometry. The experimental results revealed that the photocatalytic oxidation by the TiO2-BN nanocomposites is a multi-step process and the interactions between ibuprofen molecules and the TiO2-BN nanocomposites govern the adsorption process. The increasing BN nanosheet content in the TiO2 nanofibers facilitated the breakdown of ibuprofen degradation intermediates (hydroxyibuprofen, carboxyibuprofen, and oxypropyl ibuprofen). Kinetic modeling indicated both adsorption and photocatalytic oxidation of ibuprofen by the TiO2-BN nanocomposites followed the first-order kinetic model. The photocatalytic oxidation rate increased with the increasing BN content in the nanocomposite catalysts, which was attributed to the enhanced light absorption capacity and the separation efficiency of the photoexcited electron (e−)-hole (h+) pairs. Multiple photocatalytic cycles were conducted to investigate the reusability and regeneration of the nanofibers for ibuprofen degradation.

Applications of mass spectrometry-based targeted and non-targeted lipidomics

Recent advances in mass spectrometry have expanded our knowledge of lipids and lipid metabolic pathways involved in many (patho)physiological events. Targeted and non-targeted lipidomics are powerful analytical strategies with distinct features, and a combination of these two approaches is often employed to maximize the coverage of lipid species detected and quantified in complex biological matrices. This review briefly summarizes the applications of targeted and non-targeted lipidomics, mainly focusing on electrospray ionization–liquid chromatography–tandem mass spectrometry (ESI–LC–MS/MS), along with recent technical advances in the field. Current limitations and challenges in lipidomics and possible solutions are also discussed.

Comparison of GLA variants of unknown significance and the specific mutations causing moderate Fabry disease

Fabry disease (FD), an X-linked lysosomal storage disorder, results from the deficient activity of α-galactosidase A (α-Gal A) and the accumulation of its substrates, globotriaosylceramide (Gb3) and its deacylated derivative, globotriaosyl-sphingosine (Lyso-Gb3). Here, we compared the levels of Lyso-Gb3 in dried blood spots (DBS) and sera in affected males and heterozygotes with the “Classic” and “Later-Onset” phenotypes.The Lyso-Gb3 concentrations in DBS and sera from 56 FD patients were determined by highly sensitive electrospray ionization liquid chromatography tandem mass spectrometry.The serum Lyso-Gb3 levels in 18 and 5 affected males with the Classic and Later-Onset phenotypes, were 61 ± 38 and 14 ± 12 ng/mL, respectively. Lyso-Gb3 levels in 30 females from Classic families and three females from Later-Onset families were 10 ± 5.4 and 2.4 ± 1.0 ng/mL, respectively. The linear regression model with serum Lyso-Gb3 as the dependent variable and DBS Lyso-Gb3 an independent variable was described by the function y = − 1.83 + 1.68 ∗ x and showed a high coefficient of determination, R2 = 0.976. The overall correlation between the Lyso-Gb3 levels in DBS and sera was high (R = 0.99; p < 0.001).DBS provides a convenient, sensitive, and reproducible source to measure Lyso-Gb3 levels for diagnosis, initial phenotypic assignment, and therapeutic monitoring in patients with Fabry disease.

Paternal urinary concentrations of organophosphate flame retardant metabolites, fertility measures, and pregnancy outcomes among couples undergoing in vitro fertilization

BackgroundUse of organophosphate flame retardants (PFRs) has increased over the past decade following the phase out of some brominated flame retardants, leading to increased human exposure. We recently reported that increasing maternal PFR exposure is associated with poorer pregnancy outcomes among women from a fertility clinic. Because a small epidemiologic study previously reported an inverse association between male PFR exposures and sperm motility, we sought to examine associations of paternal urinary concentrations of PFR metabolites and their partner's pregnancy outcomes. MethodsThis analysis included 201 couples enrolled in the Environment and Reproductive Health (EARTH) prospective cohort study (2005–2015) who provided one or two urine samples per IVF cycle. In both the male and female partner, we measured five urinary PFR metabolites [bis(1,3-dichloro-2-propyl) phosphate (BDCIPP), diphenyl phosphate (DPHP), isopropylphenyl phenyl phosphate (ip-PPP), tert-butylphenyl phenyl phosphate (tb-PPP) and bis(1-chloro-2-propyl) phosphate (BCIPP)] using negative electrospray ionization liquid chromatography tandem mass spectrometry (LC-MS/MS). The sum of the molar concentrations of the urinary PFR metabolites was calculated. We used multivariable generalized linear mixed models to evaluate the association of urinary concentrations of paternal PFR metabolites with IVF outcomes, accounting for multiple in vitro fertilization (IVF) cycles per couple. Models were adjusted for year of IVF treatment cycle, primary infertility diagnosis, and maternal urinary PFR metabolites as well as paternal and maternal age, body mass index, and race/ethnicity. ResultsDetection rates were high for paternal urinary concentrations of BDCIPP (84%), DPHP (87%) and ip-PPP (76%) but low for tb-PPP (12%) and zero for BCIPP (0%). We observed a significant 12% decline in the proportion of fertilized oocytes from the first to second quartile of male urinary ΣPFR and a 47% decline in the number of best quality embryos from the first to third quartile of male urinary BDCIPP in our adjusted models. An 8% decline in fertilization was observed for the highest compared to lowest quartile of urinary BDCIPP concentrations (95% CI: 0.01, 0.12, p-trend=0.06). ConclusionsUsing IVF as a model to investigate human reproduction and pregnancy outcomes, we found that paternal urinary concentrations of BDCIPP were associated with reduced fertilization. In contrast to previously reported findings for the female partners, the paternal urinary PFR metabolites were not associated with the proportion of cycles resulting in successful implantation, clinical pregnancy, and live birth. These results indicate that paternal preconception exposure to TDCIPP may adversely impact successful oocyte fertilization, whereas female preconception exposure to ΣPFRs may be more relevant to adverse pregnancy outcomes.

An In Vivo Rat α-D-glucose Stable Isotope Homeostasis Drug Discovery Screen: A Targeted Metabolomics Approach.

We have developed a targeted metabolomics screen which consists of using two isotopically labeled glucose compounds to conduct a dual oral glucose tolerance test in rats. This dual isotopic oral glucose tolerance test (DIS-OGTT) can be used to select drug candidates that have "on"- target or have "off"-target effects on oral glucose absorption, hepatic glucose production or glucose disposal. The DIS-OGTT assay utilized intravenously administered [6-13C1-6, 6'-2H2]-glucose and orally administered [U-13C6] glucose to monitor glucose homeostasis. In the experiment, a constant intravenous dose of [6-13C1-6, 6'-2H2] glucose was converted in vivo to a series of [M+1] glucose isotopomers and unlabeled [M] glucose via gluconeogenesis while the orally administered [U-13C6] glucose was converted to a series of [M+3] and [M+2] glucose isotopomers via gluconeogenesis. The detection platform of the assay was based on a negative mode electrospray ionization liquid chromatography tandem mass spectrometry method where the deprotonated glucose anion and its various isotopomers were quantitated in rat plasma using multiple reaction monitoring techniques. The in vivo rat DIS-OGTT assay was a sensitive method for understanding drug candidates underlying postprandial effects on glucose absorption, hepatic glucose production, and insulin controlled glucose disposal. Since glucose derivatization was not required for this assay, a higher sample throughput DIS-OGTT was achieved.

Urinary Concentrations of Organophosphate Flame Retardant Metabolites and Pregnancy Outcomes among Women Undergoing in Vitro Fertilization.

Background:Evidence from animal studies suggests that exposure to organophosphate flame retardants (PFRs) can disrupt endocrine function and impair embryo development. However, no epidemiologic studies have been conducted to evaluate effects on fertility and pregnancy outcomes.Objectives:We evaluated associations between urinary concentrations of PFR metabolites and outcomes of in vitro fertilization (IVF) treatment among couples recruited from an academic fertility clinic.Methods:This analysis included 211 women enrolled in the Environment And Reproductive Health (EARTH) prospective cohort study (2005–2015) who provided one or two urine samples per IVF cycle. We measured five urinary PFR metabolites [bis(1,3-dichloro-2-propyl) phosphate (BDCIPP), diphenyl phosphate (DPHP), isopropylphenyl phenyl phosphate (ip-PPP), tert-butylphenyl phenyl phosphate (tb-PPP), and bis(1-chloro-2-propyl) phosphate (BCIPP)] using negative electrospray ionization liquid chromatography tandem mass spectrometry (LC-MS/MS). Molar concentrations of the urinary PFR metabolites were summed. We used multivariable generalized linear mixed models to evaluate the association of the PFR metabolites with IVF outcomes, accounting for multiple IVF cycles per woman.Results:Detection frequencies were high for BDCIPP (87%), DPHP (94%), and ip-PPP (80%), but low for tb-PPP (14%) and BCIPP (0%). We observed decreased success for several IVF outcomes across increasing quartiles of both summed and individual PFR metabolites (DPHP and ip-PPP) in our adjusted multivariable models. Significant declines in adjusted means from the lowest to highest quartile of were observed for the proportion of cycles resulting in successful fertilization (10% decrease), implantation (31%), clinical pregnancy (41%), and live birth (38%).Conclusions:Using IVF to investigate human reproduction and pregnancy outcomes, we found that concentrations of some urinary PFR metabolites were negatively associated with proportions of successful fertilization, implantation, clinical pregnancy, and live birth. https://doi.org/10.1289/EHP1021

Genotype, phenotype and disease severity reflected by serum LysoGb3 levels in patients with Fabry disease.

Fabry disease (FD) is a rare X-linked lysosomal storage disease caused by mutations in the α-galactosidase A (GLA) gene causing deficiency of α-galactosidase A which results in progressive glycosphingolipid accumulation, especially globotriaosylceramide (Gb3), in body liquids and lysosomes. In a large cohort of FD patients, we aimed to establish genotype/phenotype relations as indicated by serum LysoGb3 (deacylated Gb3). In 69 consecutive adult FD patients (males: n=28 (41%)) with a GLA-mutation confirmed diagnosis, we conducted a multidisciplinary clinical characterization during their routine annual examinations, and measured serum LysoGb3 levels by high-sensitive electrospray ionization liquid chromatography tandem mass spectrometry. Serum levels of LysoGb3 were significantly higher in Classic compared with Later-Onset phenotype and higher in the latter compared with controls, both in males (52 [40-83] vs 9.5 [4.5-20] vs 0.47 [0.41-0.61] ng/ml, P<0.001) and in females (9.9 [7.9-14] vs 4.9 [1.6-4.9] vs 0.41 [0.33-0.48] ng/ml, P<0.001), respectively. Multivariate linear regression analysis showed that LysoGb3 levels were independently associated with, serum creatinine (β=0.09, 95%CI 0.04-0.13, P<0.001) and the presence of cardiomyopathy (β=25, 95%CI 9.8-41, P=0.002). LysoGb3 levels were higher in males with frame-shift and nonsense mutations than in males with missense mutations (84 [72-109] vs 41 [37-52] ng/ml, P=0.002). LysoGb3 relates to disease severity, enzyme replacement response, and to the genotype severity in males. LysoGb3 supports identifying patients at risk who require intensive monitoring and treatment. LysoGb3 appears to be one marker of metabolic phenotyping of FD.

Correlation of Lyso-Gb3 levels in dried blood spots and sera from patients with classic and Later-Onset Fabry disease.

Fabry disease (FD), an X-linked lysosomal storage disorder, results from the deficient activity of α-galactosidase A (α-Gal A) and the accumulation of its substrates, globotriaosylceramide (Gb3) and its deacylated derivative, globotriaosyl-sphingosine (Lyso-Gb3). Here, we compared the levels of Lyso-Gb3 in dried blood spots (DBS) and sera in affected males and heterozygotes with the "Classic" and "Later-Onset" phenotypes. The Lyso-Gb3 concentrations in DBS and sera from 56 FD patients were determined by highly sensitive electrospray ionization liquid chromatography tandem mass spectrometry. The serum Lyso-Gb3 levels in 18 and 5 affected males with the Classic and Later-Onset phenotypes, were 61±38 and 14±12ng/mL, respectively. Lyso-Gb3 levels in 30 females from Classic families and three females from Later-Onset families were 10±5.4 and 2.4±1.0ng/mL, respectively. The linear regression model with serum Lyso-Gb3 as the dependent variable and DBS Lyso-Gb3 an independent variable was described by the function y=-1.83+1.68∗x and showed a high coefficient of determination, R2=0.976. The overall correlation between the Lyso-Gb3 levels in DBS and sera was high (R=0.99; p<0.001). DBS provides a convenient, sensitive, and reproducible source to measure Lyso-Gb3 levels for diagnosis, initial phenotypic assignment, and therapeutic monitoring in patients with Fabry disease.

Novel antiplatelet role for a protein disulfide isomerase‐targeted peptide: evidence of covalent binding to the C‐terminal CGHC redox motif

Background Protein disulfide isomerase (PDI) plays a major role in platelet aggregation, and its inhibitors have emerged as novel antithrombotic drugs. In previous work, we designed a peptide based on a PDI redox motif (CGHC) that inhibited both PDI reductase activity and PDI-modulated superoxide generation by neutrophil Nox2. Thus, we hypothesized that this peptide would also inhibit platelet aggregation by association with surface PDI. Methods Three peptides were used: CxxC, containing the PDI redox motif; Scr, presenting a scrambled sequence of the same residues and AxxA, with cysteines replaced by alanine. These peptides were tested under platelet aggregation and flow cytometry protocols to identify their possible antiplatelet activity. We labeled membrane free thiol and electrospray ionization liquid chromatography tandem mass spectrometry to test for an interaction. Results CxxC decreased platelet aggregation in a dose-dependent manner, being more potent at lower agonist concentrations, whereas neither AxxA nor Scr peptides exerted any effect. CxxC decreased aIIbb3 activation, but had no effect on the other markers. CxxC also decreased cell surface PDI pulldown without interfering with the total thiol protein content. Finally, we detected the addition of one CxxC molecule to reduced PDI through binding to Cys400 through mass spectrometry. Interestingly, CxxC did not react with oxidized PDI. Discussion CxxC has consistently shown its antiplatelet effects, both in PRP and washed platelets, corroborated by decreased aIIbb3 activation. The probable mechanism of action is through a mixed dissulphide bond with Cys400 of PDI, which has been shown to be essential for PDI's actions. Conclusion In summary, our data support antiplatelet activity for CxxC through binding to Cys400 in the PDI a0 domain, which can be further exploited as a model for sitedriven antithrombotic agent development.

Extraction efficiency and quantification of dissolved metabolites in targeted marine metabolomics

AbstractThe field of metabolomics seeks to characterize the suite of small molecules that comprise the end‐products of cellular regulation. Metabolomics has been used in biomedical applications as well as environmental studies that explore ecological and biogeochemical questions. We have developed a targeted metabolomics method using electrospray ionization–liquid chromatography tandem mass spectrometry to analyze metabolites dissolved in seawater. Preparation of samples from the marine environment presents challenges because dilute metabolites must be concentrated and desalted. We present the extraction efficiencies of 89 metabolites in our targeted method using solid phase extraction (SPE). In addition, we calculate the limits of detection and quantification for the metabolites in the method and compare the instrument response factors in five different matrices ranging from deionized water to spent medium from cultured marine microbes. High background organic matter content reduces the instrument response factor for only a small group of metabolites, yet enhances the extraction efficiency for other metabolites on the SPE cartridge used here, a modified styrene‐divinylbenzene polymer called PPL. Aromatic or larger uncharged compounds, in particular, are reproducibly well retained on the PPL polymer. This method is suitable for the detection of dissolved metabolites in marine samples, with limits of detection ranging from &lt; 1 pM to ∼ 2 nM dependent on the dual impacts of seawater matrix on extraction efficiency and on instrument response factors.

Metabolic progression to clinical phenotype in classic Fabry disease

BackgroundFabry disease is an X-linked lysosomal storage disorder due to α-galactosidase A (α-Gal A) deficiency. Clinical onset of Fabry disease is preceded by significant storage of globotriaosylceramide (Gb3) and related glycosphingolipids, but the extent of the metabolic progression before symptoms is unknown. Using a newly recognized effector and marker of Fabry disease, globotriaosylsphingosine (LysoGb3), we aimed to provide a metabolic picture of classic Fabry disease from the neonatal period to childhood.MethodsLysoGb3 was assessed at different times in two brothers with classic Fabry disease (genotype c. 370–2 A > G). The firstborn was diagnosed after clinical onset at 11 years of age, whereas the second-born was diagnosed in the neonatal period. LysoGb3 was measured in dried blood spots by high-sensitive electrospray ionization liquid chromatography tandem mass spectrometry.ResultsBlood LysoGb3 concentrations were consistent with patients’ age and clinical picture, with lower levels in the asymptomatic neonate (19.1 ng/ml) and higher levels in the symptomatic child (94.3 ng/ml). In the second-born, LysoGb3 doubled during the first 5 months of life (37.4 ng/ml), reaching ~40% concentration observed in the symptomatic period. The neonatal LysoGb3 concentration in classic Fabry disease exceeded that observed in normal subjects by over 15 times.ConclusionsA substantial increase of LysoGb3 was documented during the first months of life in classic Fabry disease, suggesting an early plateau during the pre-symptomatic period. Such a progressive metabolic trend during the pre-symptomatic period implies the potential definition of a metabolic threshold useful for a preventive therapeutic approach of classic Fabry disease. Additionally, the consistent increase of LysoGb3 in the neonatal period in classic Fabry disease suggests LysoGb3 as a useful marker for improving the specificity of newborn screening for Fabry disease.

Plasma LysoGb3: A useful biomarker for the diagnosis and treatment of Fabry disease heterozygotes.

Fabry disease (FD) is a rare X-linked lysosomal storage disorder due to mutations in the α-galactosidase A gene (GLA) that result in absent or markedly reduce α-galactosidase A (α-GalA) enzymatic activity. As a result, the major glycosphingolipid substrates, globotriaosylceramide (Gb3) and globotriaosylsphingosine (LysoGb3) accumulate in plasma, urine and tissue lysosomes. In females, the diagnosis can be complicated by the fact that 40-50% of GLA-mutation confirmed heterozygotes have normal or only slightly decreased leukocyte α-GalA activities. Recently, LysoGb3 has been appreciated as a novel FD biomarker, especially for therapeutic monitoring. Among our GLA-mutation proven FD patients, we screened 18 heterozygotes whose leukocyte α-GalA activity was determined at initial diagnosis. For these females, we measured their serum LysoGb3 levels using highly-sensitive electrospray ionization liquid chromatography tandem mass spectrometry. We identified three unrelated females in whom the accumulating LysoGb3 was increased, whereas their leukocyte α-GalA activities were in the normal range. LysoGb3 serves as an useful biomarker to improve the diagnosis of FD heterozygotes and for therapeutic evaluation and monitoring.

Golf ball-assisted electrospray ionization of mass spectrometry for the determination of trace amino acids in complex samples

During the electrospray ionization (ESI) process, ions move through a heated capillary aperture to be detected on arrival at a mass analyzer. However, the ESI process creates an ion plume, which expands into an ion cloud with an area larger than that of the heated capillary aperture, significantly contributing to an ion loss of 50% due to coulombic repulsion. The use of DC and RF fields to focus ions from the ion source into the vacuum chamber has been proposed in the literature, but the improvement of ion transmission efficiency is limited. To improve ion transmission, in this study we propose a novel method using a home-made golf ball positioned between the ion source and the inlet of the mass analyzer to hydrodynamically focus the ions passing through the golf ball. The ion plume produced by the ESI process passes through the golf ball will reduce the size of the ion cloud then be focused and most of them flowed into the mass analyzer. Therefore, the sensitivity will be improved, the aim of this investigation is to study the enhancing of the signal using golf ball-assisted electrospray ionization liquid chromatography tandem mass spectrometry (LC-MS/MS) to determine 20 trace amino acids in complex samples, including tea, urine and serum. The results showed that the analytical performance of the determination of the 20 amino acids in tea, urine and serum samples using the home-made golf ball-assisted ESI source is better than that of a commercial ESI source. The signal intensities of the 20 amino acids were enhanced by factors of 2–2700, 11–2525, and 31–342680 in oolong tea, urine and serum analyses, respectively. The precision of the proposed method ranged from 1–9%, 0.4–9% and 0.4–8% at low, medium and high concentration levels of amino acids, respectively. The home-made golf ball-assisted ESI source effectively increased the signal intensity and enhanced the ion transmission efficiency and is also an easy, convenient and economical device. This technique can be applied to the analysis of trace compounds in complex matrices.