Chiral Capillary Electrokinetic Chromatography: Principle and Applications, Detection and Identification, Design of Experiment, and Exploration of Chiral Recognition Using Molecular Modeling

This work reviews the literature of chiral capillary electrokinetic chromatography from January 2016 to March 2021. This is done to explore the state-of-the-art approach and recent developments carried out in this field. The separation principle of the technique is described and supported with simple graphical illustrations, showing migration under normal and reversed polarity modes of the separation voltage. The most relevant applications of the technique for enantioseparation of drugs and other enantiomeric molecules in different fields using chiral selectors in single, dual, or multiple systems are highlighted. Measures to improve the detection sensitivity of chiral capillary electrokinetic chromatography with UV detector are discussed, and the alternative aspects are explored, besides special emphases to hyphenation compatibility to mass spectrometry. Partial filling and counter migration techniques are described. Indirect identification of the separated enantiomers and the determination of enantiomeric migration order are mentioned. The application of Quality by Design principles to facilitate method development, optimization, and validation is presented. The elucidation and explanation of chiral recognition in molecular bases are discussed with special focus on the role of molecular modeling

Development and Validation of a Bioanalytical Method for the Quantification of Nitrated Fatty Acids in Plasma Using LC-MS/MS: Application to Cardiovascular Patients

Nitrated fatty acids (NO2-FAs) are a newly discovered class of biologically active compounds with distinct biochemical features that induce physiologically beneficial alterations in transcriptional regulatory protein function, leading to a variety of modulatory and protective actions. The most common NO2-FAs identified in vivo so far are nitro oleic acid (NO2-OA), nitro linoleic acid (NO2-LA) and its structural isomer nitro-conjugated linoleic acid (NO2-cLA). Analytical limitations that compromise accurate quantitation of these endogenous compounds are their low concentrations, compromised stability and different distribution profiles in tissues and biofluids. As a result, reliable analytical methods for the quantitative determination of their endogenous levels are rare. Only NO2-OA was quantified by GC-MS while LC-MS methods are still scarce. In this work, an LC-MS/MS bioanalytical method was developed and validated for the quantification of NO2-OA and NO2-LA in human plasma via a standard addition protocol after protein precipitation, liquid extraction and LC-MS/MS analysis in the negative ion mode. Quantification was performed via multiple reaction monitoring of the transitions m/z 326 > 46 and m/z 324 > 46 for NO2-OA and NO2-LA, respectively, and m/z 269 > 250 for the internal standard heptadecanoic acid. Linear responses were observed for both analytes over the studied range (R2 = 0.9805 and 0.9644 for NO2-OA and NO2-LA, respectively). Sufficient accuracy and precision were also achieved at low, medium and high levels within the linearity range. The limits of quantification of our method (2 nM for both NO2-FAs) were below basal endogenous levels, thereby providing a good tool to accurately measure these NO2-FAs in plasma. We applied the validated method to compare NO2-OA and NO2-LA levels in the plasma of 28 ischemic heart disease (IHD) patients and 18 healthy controls. The levels of NO2-OA were found to be significantly higher in the plasma of patients (21.7 ± 9.8 nM) versus healthy controls (12.6 ± 6 nM) (p-value < 0.01). Whereas the levels of NO2-LA were comparable in both groups (3 ± 1 nM in patients, 3.2 ± 1.7 nM in controls, p-value = 0.87288). The early elevation of NO2-OA in plasma samples, which were collected 2–3 h post myocardial injury, implies the potential use of NO2-OA levels as a biomarker for IHD after further investigation with a larger number of IHD patients. To our knowledge, this is the first comparative study on the levels of NO2-FAs in humans with and without IHD

MiR-29a promotes lipid droplet and triglyceride formation in HCV infection by inducing expression of SREBP-1c and CAV1

Aims: To examine the regulation of SREBP-1c and CAV1 by microRNA-29a (miR-29a) in cells infected with hepatitis C virus (HCV) in an attempt to control HCV-induced non-alcoholic fatty liver disease. Methods: In order to examine the manipulation of SREBP-1c and CAV1 by miR-29a, oleic acid (OA)-treated JFH-I-infected Huh-7 cells were used. OA was added 24 h post-transfection and gene expression was investigated by qRT-PCR at 48 h post treatment. The functional impact of the observed alteration in SREBP-1c and CAV1 expression was analyzed by examining lipid droplet (LD) and triglyceride (TG) content at 72 h post-OA treatment using light microscopy and spectrophotometry, respectively. Viral load was quantified by qRT-PCR at 72 h post-transfection. Results: OA treatment induced the expression of miR-29a and SREBP-1c, as compared to untreated cells. Forced miR-29a expression led to a significant up-regulation of SREBP-1c as well as CAV1 compared to mock untransfected cells. Ectopic expression of miR-29a resulted in a marked increase in LDs and their respective TGs, while miR-29a antagomirs decreased both the LD and TG content compared to mock untransfected cells. Moreover, forcing the expression of miR-29a in JFH-1 HCV-infected Huh-7 cells resulted in 53% reduction in viral titers compared to mock untransfected Huh-7 cells. Conclusion: Inducing miR-29a expression significantly induces SREBP-1c and CAV1 expression, thereby increasing LDs as well as their respective TGs. Nonetheless, forcing the expression of miR-29a resulted in reduction of HCV RNA levels in Huh-7 cells