LC Mass Research Articles

Initiation of metformin in early pregnancy results in fetal bioaccumulation, growth restriction, and renal dysmorphology in a primate model

BackgroundIn recent years, pragmatic metformin use in pregnancy has stretched to include prediabetes, type 2 diabetes, gestational diabetes and (most recently) pre-eclampsia. With its expanded use, however, concerns of unintended harm have been raised. ObjectiveWe developed an experimental primate model and applied triple-quadruple pole LC mass spectrometry (UHPLC-QQQ) for direct quantitation of maternal and fetal tissue metformin levels with detailed fetal biometry and histopathology. Study designWithin 30 days of confirmed conception (defined as early pregnancy), n=13 time-bred (TMB) Rhesus dams with gestations designated for fetal necropsy were initiated on twice daily human dose-equivalent 10 mg/kg metformin or vehicle control. Pregnant dams were maintained as pairs and fed either a control chow or 36% fat Western-style diet (WSD). Metformin or placebo vehicle control were delivered in a variety of treats while animals were separated via a slide. A Cesarean was performed at G145, and amniotic fluid and blood were collected and the fetus and placenta were delivered. The fetus was immediately necropsied by trained primate center personnel. All fetal organs were dissected, measured, sectioned, and processed per clinical standards. Fluid and tissue metformin levels were assayed using validated UHPLC-QQQ in SRM against standard curves. ResultsAmong the n=13 G145 pregnancies with fetal necropsy, n=1 dam and its fetal tissues had detectable metformin levels despite being allocated to the vehicle control group (>1 μM metformin/kg maternal weight or fetal/placental tissue), while a second fetus allocated to the vehicle control group had severe fetal growth restriction (birthweight 248.32 g, <1%) and was suspected of having a fetal congenital condition. After excluding these two fetal gestations from further analyses, 11 fetuses from dams initiated on either vehicle control (n=4, 3 female, 1 male fetuses) or 10 mg/kg metformin (n=7, 5 female, 2 male fetuses) were available for analyses. Among dams initiated on metformin by G30 (regardless of maternal diet), we observed significant bioaccumulation within the fetal kidney (0.78-6.06 μmol/kg, mean 2.48 μmol/kg) , liver (0.16-0.73 μmol/kg, mean 0.38 μmol/kg), fetal gut (0.28-1.22 μmol/kg, mean 0.70 μmol/kg), amniotic fluid (0.43-3.33 μmol/L, mean 1.88 μmol/L), placenta (0.16-1.0 μmol/kg , mean 0.50 μmol/kg) and fetal serum (0 -0.66 μmol/L , mean 0.23 μmol/L ), and fetal urine (4.1-174.1 μmol/L mean 38.5 μmol/L ), with fetal levels near biomolar equivalent to maternal levels (maternal serum 0.18-0.86 μmol/L , mean 0.46 μmol/L; maternal urine 42.6-254.0 μmol/L , mean 149.3 μmol/L). WSD feeding neither accelerated nor reduced metformin bioaccumulations in maternal or fetal serum, urine, amniotic fluid, placenta nor fetal tissues. In these 11 animals, fetal bioaccumulation of metformin was associated with less fetal skeletal muscle (57% lower cross-sectional area of gastrocnemius) and decreased liver, heart, and retroperitoneal fat masses (p<0.05), collectively driving lower delivery weight (p<0.0001) without changing the crown-rump length. Sagittal sections of fetal kidneys demonstrated delayed maturation, with disorganized glomerular generations and increased cortical thickness; this renal dysmorphology was not accompanied by structural nor functional changes indicative of renal insufficiency. ConclusionsWe demonstrate fetal bioaccumulation of metformin with associated fetal growth restriction and renal dysmorphology following maternal initiation of the drug within 30 days of conception in primates. Given these results and the prevalence of metformin use during pregnancy, additional investigation of any potential immediate and enduring effects of prenatal metformin use is warranted.

A potent subset of Mycobacterium tuberculosis glycoproteins as relevant candidates for vaccine and therapeutic target

Tuberculosis (TB) remains one of the most afflictive bacterial infections globally. In high burden TB countries, surveillance, diagnosis and treatment of drug resistant TB (RR and X/MDRTB) display a crucial public health challenge. Therefore, we need new TB vaccines; diagnostic and therapeutic strategies to briskly prevent disease promotion; reduce drug-resistant TB and protect everyone from disease. The study identified various potent membrane and cell wall M. tuberculosis glycolipoproteins that are relevant for diagnostics, drug and vaccine discovery. A M. tuberculosis Proskauer and Beck broth culture was extracted for total proteins by ammonium sulfate method. After ConA-Affinity Chromatography reputed glycoproteins were collected followed by 2DE gel electrophoresis and LC Mass spectrometry. A total of 293 glycoproteins were identified using GlycoPP and IEDB database. Probable conserved trans-membrane protein (Rv0954), LpqN (Rv0583), PPE68 (Rv3873), Phosphate-binding protein (Rv0932c), PPE61 (Rv3532) and LprA (Rv1270c), had the highest glycosylation percentage value with 13.86%, 11.84%, 11.68%, 11.1%, 10.59% and10.2%, respectively. Our study discloses several dominant glycoproteins that play roles in M. tuberculosis survival, and immunogenicity. These include glycoproteins involved in antigenicity, transport and biosynthesis of M. tuberculosis cell envelope, pathogen-host interaction and drug efflux pumps, which are considered as a feasible drug targets or TB new vaccine candidates.

Combined untargeted and targeted lipidomics approaches reveal potential biomarkers in type 2 diabetes mellitus cynomolgus monkeys

AbstractBackgroundThe significantly increasing incidence of type 2 diabetes mellitus (T2DM) over the last few decades triggers the demands of T2DM animal models to explore the pathogenesis, prevention, and therapy of the disease. The altered lipid metabolism may play an important role in the pathogenesis and progression of T2DM. However, the characterization of molecular lipid species in fasting serum related to T2DM cynomolgus monkeys is still underrecognized.MethodsUntargeted and targeted LC–mass spectrometry (MS)/MS‐based lipidomics approaches were applied to characterize and compare the fasting serum lipidomic profiles of T2DM cynomolgus monkeys and the healthy controls.ResultsMultivariate analysis revealed that 196 and 64 lipid molecules differentially expressed in serum samples using untargeted and targeted lipidomics as the comparison between the disease group and healthy group, respectively. Furthermore, the comparative analysis of differential serum lipid metabolites obtained by untargeted and targeted lipidomics approaches, four common serum lipid species (phosphatidylcholine [18:0_22:4], lysophosphatidylcholine [14:0], phosphatidylethanolamine [PE] [16:1_18:2], and PE [18:0_22:4]) were identified as potential biomarkers and all of which were found to be downregulated. By analyzing the metabolic pathway, glycerophospholipid metabolism was associated with the pathogenesis of T2DM cynomolgus monkeys.ConclusionThe study found that four downregulated serum lipid species could serve as novel potential biomarkers of T2DM cynomolgus monkeys. Glycerophospholipid metabolism was filtered out as the potential therapeutic target pathway of T2DM progression. Our results showed that the identified biomarkers may offer a novel tool for tracking disease progression and response to therapeutic interventions.

A universal surrogate matrix assay for urea measurement in clinical pharmacokinetic studies of respiratory diseases.

In pharmacokinetic studies for respiratory diseases, urea is a commonly used dilution marker for volume normalization of various biological matrices, owing to the fact that urea diffuses freely throughout the body and is minimally affected by disease states. In this study, we developed a convenient liquid chromatography-tandem mass spectrometry (LC-MS/MS) surrogate matrix assay for accurate urea quantitation in plasma, serum and epithelial lining fluid. Different mass spectrometer platforms and ionization modes were compared in parallel. The LC method and mass spectrometer parameters were comprehensively optimized to reduce interferences, to smooth the baseline and to maximize the signal-to-noise ratio. Saline was selected as the surrogate matrix, and its suitability was confirmed by good parallelism and accurate quality control sample measurements. Reliable and robust assay performance was demonstrated by precision and accuracy, dilution integrity, sensitivity, recovery and stability, all of which met bioanalysis requirements to support clinical studies. The assay performance was also verified and better understood by comparing it with a colorimetric assay and to a surrogate analyte assay. The newly developed surrogate matrix assay has the potential to be further expanded for urea quantitation in numerous physiological matrices.

High-throughput and reliable assessments of the ionization constant of monoprotic organic acids through an arginine based mixed mode HPLC

The charge state of a molecule is the single most prominent attribute ruling out its interactions with the surrounding environment. In a previous study, the retention of acids on the new Celeris™ Arginine (ARG) column was found to be predominantly driven by electrostatics and, specifically, their charge state. Therefore, we analysed 41 compounds in liquid chromatography with ultraviolet detection to study possible relationships between the analytical retention on this phase and the pKa of the acidic solutes. Highly significant relationships were observed indicating either a linear (r2 = 0.86) or a quadratic (r2= 0.89) trend. To improve the throughput of the method, this was transferred to LC mass spectrometry, allowing the analysis of a molecule every 3 mins. The developed method was found to be fast, reliable, accurate, easily automatable and simple to set up. Finally, the analytical column’s being industrially manufactured and commercially available offers broad applicability.

Maternal Vitamin D Insufficiency Early in Pregnancy Is Associated with Increased Risk of Preterm Birth in Ethnic Minority Women in Canada

BackgroundMaternal vitamin D insufficiency (plasma 25-hydroxyvitamin D [25(OH)D] <75 nmol/L) may play a role in ethnic disparities in rates of preterm and spontaneous preterm births. ObjectiveWe explored the relation between maternal plasma 25(OH)D concentration in the first trimester (8–14 wk of gestation) and the risk of preterm and spontaneous preterm births (<37 wk of gestation) by ethnicity. MethodsWe designed a case-control study that included 120 cases of preterm birth (<37 wk of gestation) and 360 term controls (≥37 wk of gestation) of singleton pregnancies from the 3D cohort, a multicenter study in 2456 pregnant women in Quebec, Canada. Plasma 25(OH)D was measured by LC–mass spectrometry. We compared the distribution of vitamin D status between cases and controls for 8 ethnic minority subgroups. We explored the association between maternal plasma 25(OH)D concentration and preterm and spontaneous preterm births with the use of splines in logistic regression by ethnicity. ResultsThe distributions of maternal vitamin D status (<50, 50–75, and >75 nmol/L) were different in preterm and spontaneous preterm birth cases compared with controls but only in women of ethnic minority (P-trend = 0.003 and 0.024, respectively). Among ethnic subgroups, sub-Saharan Africans (P-trend = 0.030) and Arab-West Asians (P-trend = 0.045) showed an inverse relation between maternal vitamin D status and the risk of preterm birth. Maternal plasma 25(OH)D concentrations of 30 nmol/L were associated with 4.05 times the risk of preterm birth in the total ethnic minority population (95% CI: 1.16, 14.12; P = 0.028) relative to participants with a concentration of 75 nmol/L. In contrast, there was no such association among nonethnic women (OR: 0.94; 95% CI: 0.48, 1.82; P = 0.85). There was no association when we considered only spontaneous preterm births in the total ethnic minority population (OR: 1.75; 95% CI: 0.39, 7.79; P = 0.46). ConclusionVitamin D insufficiency is associated with an increased risk of preterm birth in ethnic minority women in Canada.

Characterization of Solar Radiation-Induced Degradation Products of the Plant Sunscreen Sinapoyl Malate.

Agricultural activities at lower temperatures lead to lower yields due to reduced plant growth. Applying photomolecular heater agrochemicals could boost yields under these conditions, but UV-induced degradation of these compounds needs to be assessed. In this study, we employ liquid chromatography-mass spectrometry (LC-MS) coupled with infrared ion spectroscopy (IRIS) to detect and identify the degradation products generated upon simulated solar irradiation of sinapoyl malate, a proposed photomolecular heater/UV filter compound. All major irradiation-induced degradation products are identified in terms of their full molecular structure by comparing the IRIS spectra obtained after LC fractionation and mass isolation with reference IR spectra obtained from quantum-chemical calculations. In cases where physical standards are available, a direct experimental-to-experimental comparison is possible for definitive structure identification. We find that the major degradation products originate from trans-to-cis isomerization, ester cleavage, and esterification reactions of sinapoyl malate. Preliminary in silico toxicity investigations using the VEGAHUB platform suggest no significant concerns for these degradation products' human and environmental safety. The identification workflow presented here can analogously be applied to break down products from other agrochemical compounds. As the method records IR spectra with the sensitivity of LC-MS, application to agricultural samples, e.g., from field trials, is foreseen.

Development and Validation of an Innovative Analytical Approach for the Quantitation of Tris(Hydroxymethyl)Aminomethane (TRIS) in Pharmaceutical Formulations by Liquid Chromatography Tandem Mass Spectrometry.

A novel COVID-19 vaccine (BriLife®) has been developed by the Israel Institute for Biological Research (IIBR) to prevent the spread of the SARS-CoV-2 virus throughout the population in Israel. One of the components in the vaccine formulation is tris(hydroxymethyl)aminomethane (tromethamine, TRIS), a buffering agent. TRIS is a commonly used excipient in various approved parenteral medicinal products, including the mRNA COVID-19 vaccines produced by Pfizer/BioNtech and Moderna. TRIS is a hydrophilic basic compound that does not contain any chromophores/fluorophores and hence cannot be retained and detected by reverse-phase liquid chromatography (RPLC)-ultraviolet (UV)/fluorescence methods. Among the few extant methods for TRIS determination, all exhibit a lack of selectivity and/or sensitivity and require laborious sample treatment. In this study, LC−mass spectrometry (MS) with its inherent selectivity and sensitivity in the multiple reaction monitoring (MRM) mode was utilized, for the first time, as an alternative method for TRIS quantitation. Extensive validation of the developed method demonstrated suitable specificity, linearity, precision, accuracy and robustness over the investigated concentration range (1.2−4.8 mg/mL). Specifically, the R2 of the standard curve was >0.999, the recovery was >92%, and the coefficient of variance (%CV) was <12% and <6% for repeatability and intermediate precision, respectively. Moreover, the method was validated in accordance with strict Good Manufacturing Practice (GMP) guidelines. The developed method provides valuable tools that pharmaceutical companies can use for TRIS quantitation in vaccines and other pharmaceutical products.

Bioaccumulation of mycotoxins in human forensic liver and animal liver samples using a green sample treatment

The investigation of the mycotoxin bioaccumulation in human and animals is of wide relevance due to the potential toxicity associated with these secondary metabolites. This study proposes an analytical methodology consisting of salting-out liquid–liquid extraction (SALLE) followed by dispersive liquid–liquid microextraction (DLLME) for the determination of 13 mycotoxins: aflatoxins (G1, G2, B1 and B2), enniatins (A, A1, B and B1), beauvericin, HT-2 and T-2 toxins, zearalenone and deoxynivalenol, in human and animal liver. A targeted analysis was performed by high-performance liquid chromatography with tandem mass spectrometry (HPLC-MS/MS), as well as the screening of derived metabolites by ultrahigh performance LC and high-resolution mass spectrometry (UHPLC-HRMS). The proposed method was in-home validated, and trueness was verified by apparent recovery studies with values between 94 and 110 %. Furthermore, suitable linearities were obtained by the proposed method for all the mycotoxins and detection and quantification limits allow the mycotoxin monitoring at the low levels expected in biological samples. Repeatability and intermediate precision were calculated at two concentration levels, obtaining values of relative standard deviation below 9.5 %. The proposed methodology allowed to study the bioaccumulation of mycotoxins in both human and animal liver, demonstrating the presence of emergent mycotoxins in all liver samples analyzed, specifically enniatins B, B1 and beauvericin were detected with concentrations up to 4.04 µg kg−1.

A Single Aspergillus fumigatus Gene Enables Ergothioneine Biosynthesis and Secretion by Saccharomyces cerevisiae.

The naturally occurring sulphur-containing histidine derivative, ergothioneine (EGT), exhibits potent antioxidant properties and has been proposed to confer human health benefits. Although it is only produced by select fungi and prokaryotes, likely to protect against environmental stress, the GRAS organism Saccharomyces cerevisiae does not produce EGT naturally. Herein, it is demonstrated that the recombinant expression of a single gene, Aspergillus fumigatus egtA, in S. cerevisiae results in EgtA protein presence which unexpectedly confers complete EGT biosynthetic capacity. Both High Performance Liquid Chromatography (HPLC) and LC–mass spectrometry (MS) analysis were deployed to detect and confirm EGT production in S. cerevisiae. The localisation and quantification of the resultant EGT revealed a significantly (p < 0.0001) larger quantity of EGT was extracellularly present in culture supernatants than intracellularly accumulated in 96 h yeast cultures. Methionine addition to cultures improved EGT production. The additional expression of two candidate cysteine desulfurases from A. fumigatus was thought to be required to complete EGT biosynthesis, namely AFUA_2G13295 and AFUA_3G14240, termed egt2a and egt2b in this study. However, the co-expression of egtA and egt2a in S. cerevisiae resulted in a significant decrease in the observed EGT levels (p < 0.05). The AlphaFold prediction of A. fumigatus EgtA 3-Dimensional structure illuminates the bidomain structure of the enzyme and the opposing locations of both active sites. Overall, we clearly show that recombinant S. cerevisiae can biosynthesise and secrete EGT in an EgtA-dependent manner which presents a facile means of producing EGT for biotechnological and biomedical use.

Optimization of Ultrafast Proteomics Using an LC-Quadrupole-Orbitrap Mass Spectrometer with Data-Independent Acquisition.

Proteomics has become an increasingly important tool in medical and medicinal applications. It is necessary to improve the analytical throughput for these applications, particularly in large-scale drug screening to enable measurement of a large number of samples. In this study, we aimed to establish an ultrafast proteomic method based on 5-min gradient LC and quadrupole-Orbitrap mass spectrometer (Q-Orbitrap MS). We precisely optimized data-independent acquisition (DIA) parameters for 5-min gradient LC and reached a depth of >5000 and 4200 proteins from 1000 and 31.25 ng of HEK293T cell digest in a single-shot run, respectively. The throughput of our method enabled the measurement of approximately 80 samples/day, including sample loading, column equilibration, and wash running time. We demonstrated that our method is applicable for the screening of chemical responsivity via a cell stimulation assay. These data show that our method enables the capture of biological alterations in proteomic profiles with high sensitivity, suggesting the possibility of large-scale screening of chemical responsivity.

Isolation of Alkaloids from Papaver rhoeas (Papaveraceae) Wildly Grown in Iraq

The plant Papaver rhoeas ,which belongs to family Papaveraceae and known as common poppy is wildly grown in Iraq .It was used in traditional medicine in wide range of diseases including inflammation, diarrhea, sleep disorders, treatment of cough, analgesia, and also to reduce the withdrawal signs of opioid addiction.&#x0D; The project provide the first comprehensive research done in Iraq to study the phytochemical and the methods of extraction and separation of alkaloids from Papaver rhoeas wildly grown in Iraq .The plant was harvested in April 2019 from Zurbatiya is an Iraqi town located at the northeast of Waist province in Iraq.The collected plant was washed thoroughly, dries under shade, and grounding in a mechanical grinder to fine powder. The plant was extracted by hot extraction method using Methanol then fractionation was done to separate alkaloids from chloroform Fraction by TLC and PTLC .The alkaloids were isolated and purified by PTLC then subjected to various analytical techniques for alkaloids identification such as UV, LC mass and IR .The result was indicated of three alkaloids (dihydrocodien, chelidonine and papaverrubine C) in Papaver rhoeas plant.

Comparative study on Extraction of glucan nanoparticles from different two edible mushrooms

It was recently announced that β-glucan with globular small particles size has higher disease resistant capacity due to its induced strong immune activity. Herein, this work was designed to extract the nanoglucans (NGs) by a direct way from mushrooms using acid base procedure. Lentinula edodes (Shiitake) and Pleurotus ostreatus (oyster) mushrooms were used for production of NGs-s and NGs-o, respectively, through boiling with 0.5M NaOH for 24h. The extracted NGs were then characterized using the world-wide tools including LC-Ms, H1NMR, FTIR, SEM, TEM, DLS and UV-Visible spectra. Results from LC mass, proton NMR and FTIR as well as UV-vis analysis affirmed the chemical structure of the obtained β D-glucan in NGs compared with standard β -glucan as a reference. In addition, morphological characteristics of NGs were obtained using SEM and TEM. SEM revealed the porosity of surface with homogeneous distribution particularly in case of NGs-s. TEM also proved the formation of NGs with size ranging from 10-25 nm and 40-50nm for NGs-s and NGs-o, respectively, with needle edge shapes as in NGs-s. However, NGs-o showed a loosely aggregated irregular shapes which may attributed to their low zeta potential. This novel approach opens up opportunities for harnessing mushrooms in preparation of new cost-effective bioactive products.

Lamellar Enzyme-Metal–Organic Framework Composites Enable Catalysis on Large Substrates

Lamellar Enzyme-Metal–Organic Framework Composites Enable Catalysis on Large Substrates

Biosorption of Co (II) ions from aqueous solutions using selected local Luffa Cylindrica: Adsorption and characterization studies

The present study is concerned with the removal of Cobalt (II) from aqueous solution by adsorption onto low cost adsorbent. Luffa Cylindrica as adsorbent, (LC) was investigated in batch adsorption system. LC was characterized by Scanning Electron Microscopy (SEM) and Brunauer-Emmett-Teller (BET) surface area analyzer. The sorption of Co (II) ions by LC was subjected to equilibrium, thermodynamics and kinetic studies and was carried out by considering the effects of pH, effect of masse and particles size of the biosorbent, initial metal ions concentration, contact time and temperature. BET surface area of LC was 46.396 m 2 /g. The optimum conditions for maximum adsorption were attained at pH 6 and LC mass is 1.5 g with particle size is < 0.08 mm and contact time is 60 min. The pseudo-second-order rate equation described the Kinetic data well. The process is chemisorptive and controlled by the pseudo-second-order. Adsorption parameters were determined using both Langmuir and Freundlich isotherms, but the experimental data were better fitted to the Langmuir equation than to Freundlich equation, with correlation coefficients above 0.99 which indicates the adsorption is monolayer adsorption with maximum adsorption capacity for Co(II) was 2.53 mg/g.

The synthesis and investigation of the catalytic properties of symmetric Schiff‐base ligand metal complexes

An ongoing project investigating the synthesis and catalytic properties of symmetric heterometallic Schiff‐base ligand complexes has been instigated. A novel symmetric Schiff‐Base ligand metal complex has been synthesized, characterized through IR spectroscopy, NMR spectroscopy, and LC mass spectrometry. The catalytic activity of this Schiff‐Base ligand has been investigated using cyclic voltammetry, mass spectrometric study, and UV‐vis spectroscopy. With the long‐term goal being a publication of results, this study aims to contribute to the fields of inorganic and biochemistry.Support or Funding InformationBarry Goldwater Scholarship Foundation

Obscurin and Clusterin Elevation in Serum of Acute Myocardial Infarction Patients

We discovered new biomarker candidates for acute myocardial infarction (AMI) using micro LC and accurate mass spectrometry. Two serum samples were pooled and used for biomarker screening. UHPLC‐LTQ‐Orbitrap analysis was used with data‐dependent acquisition followed by Sequest search. Six biomarker candidates were selected after fold change analysis of peptides in AMI patient samples compared to healthy controls. Literature search results were reflected. The six biomarker candidates were characterized and quantified using multi reaction monitoring using synthesized peptides or trypsin digested protein standards. Levels of obscurin, alpha‐1‐acid glycoprotein 1, and clusterin were significantly increased (p &lt; 0.001) in AMI patient samples compared to levels in healthy controls, while those of antithrombin III and fibrinogen beta were not. We confirmed the diagnostic performance of obscurin and clusterin as biomarker candidates for AMI using commercial ELISA kits. Receiver operating characteristic results showed that obscurin and clusterin showed highest sensitivity and specificity for AMI diagnosis together with troponins by both of multi‐reaction monitoring analysis and ELISA.

Validation according to European and American regulatory agencies guidelines of an LC-MS/MS method for the quantification of free and total ropivacaine in human plasma.

Background Ropivacaine is a widely used local anaesthetic drug, highly bound to plasma proteins with a free plasma fraction of about 5%. Therefore, the monitoring of free drug concentration is most relevant to perform pharmacokinetic studies and to understand the drug pharmacokinetic/pharmacodynamic (PK/PD) relationship. Methods A high-sensitivity liquid chromatography-tandem mass spectrometry (LC-MS/MS) method using reverse-phase LC and electrospray ionisation mass spectrometry with multiple reaction monitoring (MRM) is described for the quantitation of both free and total ropivacaine in human plasma. Ropivacaine-d7 was used as an internal standard (IS). Results The method was validated in the range 0.5-3000 ng/mL, with five levels of QC samples and according to the European Medicine Agency and Food and Drug Administration guidelines. The performance of the method was excellent with a precision in the range 6.2%-14.7%, an accuracy between 93.6% and 113.7% and a coefficient of variation (CV) of the IS-normalised matrix factor below 15%. This suitability of the method for the quantification of free and total ropivacaine in clinical samples was demonstrated with the analysis of samples from patients undergoing knee arthroplasty and receiving a local ropivacaine infiltration. Conclusions A method was developed and validated for the quantification of free and total ropivacaine in human plasma and was shown suitable for the analysis of clinical samples.

Neonatal adrenal insufficiency: Turkish Neonatal and Pediatric Endocrinology and Diabetes Societies consensus report.

It is difficult to make a diagnosis of adrenal insufficiency in the newborn, because the clinical findings are not specific and the normal serum cortisol level is far lower compared to children and adults. However, dehydratation, hyperpigmentation, hypoglycemia, hyponatremia, hyperkalemia and metabolic acidosis should suggest the diagnosis of adrenal insufficiency. Hypotension which does not respond to vasopressors should especially be considered a warning. If the adrenocorticotropin hormone level measured simultaneously with a low serum cortisol level is 2-fold higher than the upper normal limit of the reference range, a diagnosis of primary adrenal insufficiency is definite. Even if the serum cortisol level is normal, a diagnosis of relative adrenal insufficiency can be made with clinical findings, if the patient is under heavy stress. The serum cortisol level should be measured using the method of ‘high pressure liquid chromatography’ or ‘LC mass spectrometry’. Adrenal steroid biosynthesis can be evaluated more specifically and sensitively with ‘steroid profiling’. Rennin and aldosterone levels may be measured in addition to serum electrolytes for the diagnosis of mineralocorticoid insufficiency. Adrenocorticotropic hormone stimulation test may be used to confirm the diagnosis and elucidate the etiology. In suspicious cases, treatment can be initiated without waiting for the adrenocorticotropic hormone stimulation test. In schock which does not respond to vasopressors, intravenous hydrocortisone at a dose of 50-100 mg/m2 or a glucocorticoid drug at an equivalent dose should be initiated. In maintanence treatment, the physiological secretion rate of hydrocortisone is 6 mg/m2/day (15 mg/m2/day in the newborn). The replacement dose should be adjusted with clinical follow-up and by monitoring growth rate, weight gain and blood pressure. Fludrocortisone (0,1 mg tablet) is given for mineralocorticoid treatment (2x0,5-1 tablets). A higher dose may be needed in the neonatal period and in patients with aldosterone resistance. If hyponatremia persists, oral NACl may be added to treatment. In the long-term follow-up, patients should carry an identification card and the glucocorticoid dose should be increased 3-10-fold in cases of stress.

Design and Synthesis of A PD-1 Binding Peptide and Evaluation of Its Anti-Tumor Activity.

Immune-checkpoint blockades, suchas PD-1 monoclonal antibodies, have shown new promising avenues to treat cancers. Failure responsesof many cancer patients to these agents have led to a massive need for alternative strategies to optimize tumor immunotherapy. Currently, new therapeutic developments involve peptide blocking strategies, as they have high stability and low immunogenicity. Here, we have designed and synthesized a new peptide FITC-YT-16 to target PD-1. We have studied FITC-YT-16 by various experiments, including Molecular Operating Environment MOE modeling, purification testing by HPLC and LC mass, peptide/PD-1 conjugation and affinity by microscale thermophoresis (MST), and T cell immune-fluorescence imaging by fluorescence microscopy and flow cytometry. The peptide was tested for its ability to enhanceT cell activity against tumor cell lines, including TE-13, A549, and MDA-MB-231. Lastly, we assessed T cell cytotoxicity under peptide treatment. YT-16–PD-1 interaction showed a high binding affinity as a low energy complex that was confirmed by MOE. Furthermore, the peptide purity and molecular weights were 90.96% and 2344.66, respectively. MST revealed that FITC-YT-16 interacted with PD-1 at a Kd value of 17.8 ± 2.6 nM. T cell imaging and flow cytometry revealed high affinity of FITC-YT-16 to PD-1. Interestingly, FITC-YT-16 efficiently blocked PD-1 signaling pathways and promoted T cell inflammatory responses by elevating IL-2 and INF-γ levels. Moreover, FITC-YT-16 has the ability to activate T cell cytotoxicity. Therefore, FITC-YT-16 significantly enhanced T cell anti-tumor activity by blocking PD-1–PD-L1 interactions.