Capillary electrophoresis-mass spectrometry as a tool for the noninvasive target metabolomic analysis of underivatized amino acids for evaluating embryo viability in assisted reproduction.

Monitoring metabolite uptake and excretion in the culture medium is a noninvasive technique that is used for the metabolic study of cleaving embryos after in vitro fertilization. Low sample consumption, the versatility of the detection, and optimal sensitivity and selectivity are essential elements for extracellular metabolome analyses, and can be conveniently achieved by combining CE with mass spectrometric detection. This paper reports a method for amino acid determination in a limited volume sample (8 µL) of spent culture media collected after the cultivation of in vitro fertilized embryos. Special attention was focused on the sample preparation procedure. The sample was processed with acetonitrile, which facilitates online sample preconcentration via field-amplified sample stacking, and undesired sample evaporation was significantly reduced by the simultaneous addition of dimethyl sulfoxide. Key parameters that affected electrophoretic separation and mass spectrometric detection were investigated, including the type of buffers and organic solvent, optimization of their concentrations, and finally the settings for their ionization. The separation and quantification of 19 amino acids were achieved using 15% acetic acid as the background electrolyte with a sheath liquid consisting of an equimolar mixture of methanol and water. The applicability of the optimized system was demonstrated by determining the amino acid profile in 40 samples of spent cultivation medium in this pilot study. This developed method also has great potential for amino acid analyses in minute sample volumes of other biological matrices.

Hyaluronidase reaction kinetics evaluated by capillary electrophoresis with UV and high-resolution mass spectrometry (HRMS) detection.

The biology of hyaluronidase activity on age related turnover of the hyaluronic acid (HA) in skin dermis and epidermis has not been established. Elucidation of this phenomenon enables discovery of novel compounds for skin health. As a simple and green technique, capillary electrophoresis (CE) was used for the first time for the determination of the kinetic constants (Km, Vmax and IC50) of the enzymatic degradation of HA. Reaction products were identified using CE/high-resolution mass spectrometry (HRMS) after appropriate optimization. Best results in terms of signal sensitivity were obtained using 10 mM ammonium acetate (pH 9.0) BGE, a sheath liquid composed of methanol-water (80:20, v/v) with 0.02% (v/v) formic acid at 10 µL min-1 and an ESI voltage at -4 kV. Km and Vmax were determined (n = 3) using CE/UV at 200 nm as 0.24 ± 0.02 mg mL-1 and 150.4 ± 0.1 nM s-1, respectively. They were also successfully obtained by CE/HRMS (n = 3) with Km of 0.49 ± 0.02 mg mL-1 and Vmax of 155.7 ± 0.2 nM s-1. IC50 of a standard natural inhibitor, epigallocatechin gallate, was also determined by CE-UV/HRMS. Kinetic constant values obtained by CE compared well with literature which validated the developed CE-based assay. In addition, the activity of homemade tetrasaccharides of biotinylated chondroitin sulfate CS-A or CS-C (4- or 6- sulfated in a homogeneous or heterogeneous way) on the hydrolysis reaction of hyaluronidase was evaluated. Hyaluronidase was mostly dose-dependently inhibited by CS-A tetrasaccharides sulfated in a homogeneous way. Two trisaccharides from truncated linkage region of proteoglycans were also tested as inhibitors or activators. CE-based assay showed that even a small modification of one hydroxyl group changes the influence on hyaluronidase activity. CE-based assay can be used for the screening of natural and synthetic inhibitors of hyaluronidase activity for cosmetic and therapeutic applications.

Combination of on-line CE assay with MS detection for the study of drug metabolism by cytochromes P450.

A new CE-MS method with enzymatic reaction inside the capillary was developed for the study of drug metabolism by cytochromes P450. This automated method, based on the transverse diffusion of laminar flow profiles methodology, is comprised of the injection of substrates and enzyme, their mixing, incubation, and separation of the reaction products, all performed by CE, and their detection, identification, and quantification by MS. The developed and validated method was finally used to conduct a kinetic study of cytochrome P450 isoform 2C9 or human liver microsomes with diclofenac in order to demonstrate its practical functionality. All the estimated kinetic values--apparent Michaelis constants and apparent maximum reaction velocities were in agreement with literature data obtained using other techniques. In addition, the consumption of reactants was in the tens of nL per analysis. The method's usability was further demonstrated on tolbutamide, the other probe substrate of cytochrome P450 isoform 2C9. As a result, the method is conceptually applicable for the screening of any other cytochrome P450 isoform and its substrates and inhibitors after adapting the incubation and separation conditions.

Nonaqueous capillary electrophoresis of dextromethorphan and its metabolites.

This study deals with the nonaqueous capillary electrophoretic separation of dextromethorphan and its metabolites using a methanolic background electrolyte. The optimization of separation conditions was performed in terms of the resolution of dextromethorphan and dextrorphan and the effect of separation temperature, voltage, and the characteristics of the background electrolyte were studied. Complete separation of all analytes was achieved in 40 mM ammonium acetate dissolved in methanol. Hydrodynamic injection was performed at 3 kPa for 4 s. The separation voltage was 20 kV accompanied by a low electric current. The ultraviolet detection was performed at 214 nm, the temperature of the capillary was 25°C. These conditions enabled the separation of four analytes plus the internal standard within 9 min. Further, the developed method was validated in terms of linearity, sensitivity, and repeatability. Rat liver perfusate samples were subjected to the nonaqueous capillary electrophoretic method to illustrate its applicability.

New capillary electrophoretic method for on-line screenings of drug metabolism mediated by cytochrome P450 enzymes.

A new method for the determination of kinetic and inhibition parameters of cytochromes P450 reactions by means of on-line CE was developed. It is based on transverse diffusion of laminar flow profiles methodology introduced by Krylov et al. that injection procedure was modified. The solutions of an enzyme and its substrates are injected by hydrodynamic pressure as a series of repeated consecutive plugs. Proposed injection of three plugs of enzyme surrounded with plugs of substrates represents a certain trade-off to obtain the reaction mixture with the satisfying homogeneity by the short-injection procedure as possible. Mathematical modeling confirmed the assumption of a consistent distribution of reactants in the final reaction mixture. Kinetic and inhibition studies of cytochrome P450 isoform 2C9's reaction with diclofenac as a probe substrate and sulfaphenazole as a probe inhibitor were conducted in order to prove the practical applicability of the proposed method for on-line screenings of drug metabolism mediated by cytochrome P450 enzymes. As a result, an apparent Michaelis constant of 2.66 ± 0.18 µM, apparent maximum reaction velocity of 7.91 ± 0.22 nmol min(-1) nmol(-1) , Hill coefficient of 1.59 ± 0.16, half maximal inhibitory concentration of 0.94 ± 0.04 µM and apparent inhibition constant of 0.39 ± 0.07 µM were determined. All these values are in agreement with literature data obtained using different techniques. In addition, less than 30 nL of cytochrome P450 2C9 solution was consumed per analysis in the kinetic and inhibition studies using this method.