Fast, sensitive LC-MS resolution of α -hydroxy acid biomarkers via SPP-teicoplanin and an alternative UV detection approach.

Enantioseparation of α -hydroxy acids is essential since specific enantiomers of these compounds can be used as disease biomarkers for diagnosis and prognosis of cancer, brain diseases, kidney diseases, diabetes, etc., as well as in the food industry to ensure quality. HPLC methods were developed for the enantioselective separation of 11 α -hydroxy acids using a superficially porous particle-based teicoplanin (TeicoShell) chiral stationary phase. The retention behaviors observed for the hydroxy acids were HILIC, reversed phase, and ion-exclusion. While both mass spectrometry and UV spectroscopy detection methods could be used, specific mobile phases containing ammonium formate and potassium dihydrogen phosphate, respectively, were necessary with each approach. The LC-MS mode was approximately two orders of magnitude more sensitive than UV detection. Mobile phase acidity and ionic strength significantly affected enantioresolution and enantioselectivity. Interestingly, higher ionic strength resulted in increased retention and enantioresolution. It was noticed that for formate-containing mobile phases, using acetonitrile as the organic modifier usually resulted in greater enantioresolution compared to methanol. However, sometimes using acetonitrile with high ammonium formate concentrations led to lengthy retention times which could be avoided by using methanol as the organic modifier. Additionally, the enantiomeric purities of single enantiomer standards were determined and it was shown that almost all standards contained some levels of enantiomeric impurities.

Effect of position of deuterium atoms on gas chromatographic isotope effects.

Deuterium substitution provides various benefits in drug molecules, including improvement in pharmacokinetic properties, reduction of toxicity, reduction of epimerization, etc. Also, it has been shown that the position of deuterium substitution affects the properties of drug molecules. Therefore, it is important to study low molecular weight deuterated isotopologues which constitute the deuterated pool and are building blocks of larger deuterated molecules. The effect of the position and number of deuterium atoms on the retention of 23 deuterated isotopologues on two gas chromatography stationary phases of different polarities was evaluated. It was observed that the ratio of calculated chromatographic isotope effects resulting from a deuterium atom connected to an sp2 vs. an sp3 hybridized carbon was more on the polar IL-111i stationary phase compared to the nonpolar PDMS-5, for each group of isotopologues. Also, a compound with a deuterium atom connected to an sp2 hybridized carbon always had greater retention than the analogous compound where deuterium was connected to an sp3 hybridized carbon. The van't Hoff plots for all analytes showed that the effect of entropy was almost negligible in the separation of deuterated vs. protiated isotopologues, thus these separations were mainly enthalpy driven.

High information spectroscopic detection techniques for gas chromatography.

Gas chromatography has always been a simple and widely used technique for the separation of volatile compounds and their quantitation. However, the common detectors used with this technique are mostly universal and do not provide any specific qualitative information. There have been some attempts to combine the separation power of GC with the qualitative capabilities of "high-information" spectroscopic techniques including infrared spectroscopy, nuclear magnetic resonance spectroscopy, molecular rotational resonance spectroscopy, and vacuum ultraviolet spectroscopy. Some of these hyphenations have proven to be quite successful while others were less so. The history of such attempts, up to the most recent studies in this area, are discussed. Most recently, the hyphenation of GC with molecular rotational resonance spectroscopy which provides promising results and is a newly developed technique is reviewed and compared to previous high-information spectroscopic detection approaches. The history, description and features of each method along with their applications and challenges are discussed.

An examination of the effects of water on normal phase enantioseparations.

Superficially porous silica bonded with macrocyclic glycopeptides can separate enantiomers in various chromatographic formats, including normal phase liquid chromatography (NPLC). The conventional wisdom in NPLC is to avoid intentionally adding water in the eluents. Herein we examine the effects of small quantities of water as an additive on chiral separations in NPLC with the n-hexane-ethanol system. A phase diagram (n-hexane-ethanol-water) is used to analyze the physicochemical properties of the mobile phase. The relative polarity change of solvents upon adding water was determined by using bathochromic shifts of dissolved Nile Red dye. The effectiveness of chiral NPLC with water traces is demonstrated for various pharmaceutically relevant enantiomeric compounds. It is postulated that water molecules weaken stationary phase-solute interactions, resulting in lower retention times for both enantiomers in addition to significantly higher efficiencies. Gibbs free energy changes provided an understanding of the different enantioselectivity shifts caused by water addition. Some interesting kinetic effects also were observed. Classical van Deemter curves are not observed on macrocyclic glycopeptide stationary phases due to slow mass transfer kinetics and thermal effects at high flow rates. The most significant advantage of adding water in NPLC is reducing mass transfer kinetics and altering the mass overloading properties which is highly beneficial on macrocyclic glycopeptide phases. By overloading a 10 × 0.46-cm column with up to 0.6 mg alprenolol, it was found that the relative adsorption isotherm of the first eluting enantiomer was switched from Langmuir to anti-Langmuir type by water addition. The peak shape tuning effect demonstrated the strong influence of water on specific interaction sites of the chiral stationary phases. Water addition effects were most beneficial for enantiomeric and preparative separations in NP mode.

Hydrogel Mechanics Influence the Growth and Development of Embedded Brain Organoids.

Brain organoids are three-dimensional, tissue-engineered neural models derived from induced pluripotent stem cells that enable studies of neurodevelopmental and disease processes. Mechanical properties of the microenvironment are known to be critical parameters in tissue engineering, but the mechanical consequences of the encapsulating matrix on brain organoid growth and development remain undefined. Here, Matrigel was modified with an interpenetrating network (IPN) of alginate, to tune the mechanical properties of the encapsulating matrix. Brain organoids grown in IPNs were viable, with the characteristic formation of neuroepithelial buds. However, organoid growth was significantly restricted in the stiffest matrix tested. Moreover, stiffer matrixes skewed cell populations toward mature neuronal phenotypes, with fewer and smaller neural rosettes. These findings demonstrate that the mechanics of the culture environment are important parameters in brain organoid development and show that the self-organizing capacity and subsequent architecture of brain organoids can be modulated by forces arising from growth-induced compression of the surrounding matrix. This study therefore suggests that carefully designing the mechanical properties of organoid encapsulation materials is a potential strategy to direct organoid growth and maturation toward desired structures.

Enhancing Sensitivity for High-Selectivity Gas Chromatography-Molecular Rotational Resonance Spectroscopy.

A next-generation gas chromatograph-molecular rotational resonance (MRR) spectrometer (GC-MRR) with instrumental improvements and higher sensitivity is described. MRR serves as a structural information-rich detector for GC with extremely narrow linewidths and capabilities surpassing 1H nuclear magnetic resonance/Fourier transform infrared spectroscopy/mass spectrometry (MS) while offering unparalleled specificity in regard to a molecule's three-dimensional structure. With a Fabry-Pérot cavity and a supersonic jet incorporated into a GC-MRR, dramatic improvements in sensitivity for molecules up to 244 Da were achieved in the microwave region compared to the only prior work, which demonstrated the GC-MRR idea for the first time with millimeter waves. The supersonic jet cools the analytes to ∼2 K, resulting in a limited number of molecular rotational and vibrational levels and enabling us to obtain stronger GC-MRR signals. This has allowed the limits of detection of the GC-MRR to be comparable to a GC thermal conductivity detector with an optimized choice of gases. The performance of this GC-MRR system is reported for a range of molecules with permanent dipole moments, including alcohols, nitrogen heterocyclics, halogenated compounds, dioxins, and nitro compounds in the molecular mass range of 46-244 Da. The lowest amount of any substance yet detected by MRR in terms of mass is reported in this work. A theoretically unexpected finding is reported for the first time about the effect of the GC carrier gas (He, Ne, and N2) on the sensitivity of the analysis in the presence of the gas driving the supersonic jet (He, Ne, and N2) in the GC-MRR. Finally, the idea of total molecule monitoring in the GC-MRR analogous to selected ion monitoring in GC-MS is illustrated. Structural isomers and isotopologues of bromobutanes and bromonitrobenzenes are used to demonstrate this concept.

Evaluation of gas chromatography for the separation of a broad range of isotopic compounds.

The separation of deuterated compounds from their protiated counterparts is essential in areas of drug discovery and development, investigating kinetic isotope effects and quantitative methods of non-mass spectrometry-based stable isotope dilution assay (non-MS SIDA). The separations of 47 isotopologue pairs of common compounds and drugs were achieved by gas-liquid chromatography, employing twelve different stationary phases. Polydimethylsiloxane phase, phenyl substituted polydimethylsiloxane phases, wax phases, ionic liquid phases, and chiral stationary phases were selected to encompass a wide polarity range and diverse chemical interactions. The best-performing stationary phases are presented for separation of protic-polar, aprotic-dipolar, nonpolar analytes. Overall, the IL111i, SPB-20, and PAG stationary phases were remarkable in their ability to separate the isotopologues. The isotope effect was also evaluated. It was observed that nonpolar stationary phases often exhibit an inverse isotope effect in which heavier isotopic compounds elute earlier than their lighter counterparts. Conversely, polar stationary phases often show a normal isotope effect, while those of intermediate polarities can show both effects depending on the isotopologues. The location of deuterium atoms, however, affects isotopologue retention times. Deuterium substituted aliphatic groups appear to have a greater inverse isotope effect on retention than aromatic substituents.

The applications of heparin in vascular tissue engineering.

Cardiovascular diseases, among all diseases, are taking the most victims worldwide. Coronary artery occlusion, takes responsibility of about 30% of the yearly global deaths in the world (Heart Disease and Stroke Statistics 2017 At-a-Glance, 2017), raising the need for viable substitutes for cardiovascular tissues. Depending on a number of factors, blocked coronary arteries are now being replaced by autografts or stents. Since the autografts, as the gold standard coronary artery replacements, are not available in adequate quality and quantity, the demand for small diameter vascular substitute comparable to native vessels is rapidly growing. Synthetic grafts have been successfully approved for developing vascular replacements but regarding the special conditions in small-caliber vessels, their use is limited to large-diameter vascular tissue engineering. The major problems associated with the vascular tissue engineered grafts are thrombosis and intimal hyperplasia. Heparin, a negatively charged natural polysaccharide has been used in fabricating vascular grafts since it prevents protein fouling on the surfaces and most importantly, impeding thrombosis. Herein, we focused on heparin, as a multifunctional bioactive molecule that not only serves as an anticoagulant with frequent clinical use but also acts as an anti-inflammatory and angiogenic regulatory substance. We summarized heparin incorporation into stents and grafts and their applicability to restrain restenosis. Also, the applications of heparinzation of biomaterials and heparin mimetic polymers and different approaches invoked to improve heparin bioactivity have been reviewed. We summarized the methods of adding heparin to matrices as they were explained in the literature. We reviewed how heparin influences the biocompatibility of the scaffolds and discussed new advances about using heparin in small-diameter vascular tissue engineering.

Vascular tissue engineering: Fabrication and characterization of acetylsalicylic acid-loaded electrospun scaffolds coated with amniotic membrane lysate.

As the incidence of small-diameter vascular graft (SDVG) occlusion is considerably high, a great amount of research is focused on constructing a more biocompatible graft. The absence of a biocompatible surface in the lumen of the engineered grafts that can support confluent lining with endothelial cells (ECs) can cause thrombosis and graft failure. Blood clot formation is mainly because of the lack of an integrated endothelium. The most effective approach to combat this problem would be using natural extracellular matrix constituents as a mimic of endothelial basement membrane along with applying anticoagulant agents to provide local antithrombotic effects. In this study, we fabricated aligned and random electrospun poly-L-lactic acid (PLLA) scaffolds containing acetylsalicylic acid (ASA) as the anticoagulation agent and surface coated them with amniotic membrane (AM) lysate. Vascular scaffolds were structurally and mechanically characterized and assessed for cyto- and hemocompatibility and their ability to support endothelial differentiation was examined. All the scaffolds showed appropriate tensile strength as expected for vascular grafts. Lack of cytotoxicity, cellular attachment, growth, and infiltration were proved using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and scanning electron microscopy. The blood compatibilities of different scaffolds examined by in vitro hemolysis and blood coagulation assays elucidated the excellent hemocompatibility of our novel AM-coated ASA-loaded nanofibers. Drug-loaded scaffolds showed a sustained release profile of ASA in 7 days. AM-coated electrospun PLLA fibers showed enhanced cytocompatibility for human umbilical vein ECs, making a confluent endothelial-like lining. In addition, AM lysate-coated ASA-PLLA-aligned scaffold proved to support endothelial differentiation of Wharton's jelly-derived mesenchymal stem cells. Our results together indicated that AM lysate-coated ASA releasing scaffolds have promising potentials for development of a biocompatible SDVG.