On‐line two‐dimensional LC: A rapid and efficient method for the determination of enantiomeric excess in reaction mixtures

A novel LC‐based method for the determination of enantiomeric excess (ee) in a sample mixture has been developed by employing on‐line 2D LC. The orthogonal separation system is composed of an NH₂ column as the first dimension to elute the target chiral compound from the crude mixture and a chiral column as the second dimension to determine the ee of the target chiral product. A series of crude mixtures from asymmetric reactions have been directly analyzed without prepurification. Good reproducibility (intra‐ and interday precisions were all under 1.33%) and good accuracy (deviations from ee values determined by 1D HPLC were all <1.03%) have been obtained. Compared with the traditional method for the determination of ee, on‐line 2D HPLC can be used in real time and holds great potential in the time‐saving determination of ee in asymmetric synthesis.     

Evaluation of fritless solid‐phase extraction coupled on‐line with capillary electrophoresis‐mass spectrometry for the analysis of opioid peptides in cerebrospinal fluid

Fritless SPE on‐line coupled to CE with UV and MS detection (SPE‐CE‐UV and SPE‐CE‐MS) was evaluated for the analysis of opioid peptides. A microcartridge of 150 μm id was packed with a C₁₈ sorbent (particle size > 50 μm), which was retained between a short inlet capillary and a separation capillary (50 μm id). Several experimental parameters were optimized by SPE‐CE‐UV using solutions of dynorphin A (DynA), endomorphin 1 (End1), and methionine‐enkephaline (Met). A microcartridge length of 4 mm was selected, sample was loaded for 10 min at 930 mbar and the retained peptides were eluted with 67 nL of an acidic hydro‐organic solution. Using SPE‐CE‐MS, peak area and migration time repeatabilities for the three opioid peptides were 12–27% and 4–5%, respectively. SPE recovery was lower for the less hydrophobic DynA (22%) than for End1 (66%) and Met (78%) and linearity was satisfactory in all cases between 5 and 60 ng/mL. The LODs varied between 0.5 and 1.0 ng/mL which represent an enhancement of two orders of magnitude when compared with CE‐MS. Cerebrospinal fluid (CSF) samples spiked with the opioid peptides were analyzed to demonstrate the applicability to biological samples. Peak area and migration time repeatabilities were similar to the standard solutions and the opioid peptides could be detected down to 1.0 ng/mL.     

Anionic microemulsion to solvent stacking for on‐line sample concentration of cationic analytes in capillary electrophoresis

The common SDS microemulsion (i.e. 3.3% SDS, 0.8% octane, and 6.6% butanol) and organic solvents were investigated for the stacking of cationic drugs in capillary zone electrophoresis using a low pH separation electrolyte. The sample was prepared in the acidic microemulsion and a high percentage of organic solvent was included in the electrolyte at anodic end of capillary. The stacking mechanism was similar to micelle to solvent stacking where the micelles were replaced by the microemulsion for the transport of analytes to the organic solvent rich boundary. This boundary is found between the microemulsion and anodic electrolyte. The effective electrophoretic mobility of the cations reversed from the direction of the anode in the microemulsion to the cathode in the boundary. Microemulsion to solvent stacking was successfully achieved with 40% ACN in the anodic electrolyte and hydrodynamic sample injection of 21 s at 1000 mbar (equivalent to 30% of the effective length). The sensitivity enhancement factors in terms of peak height and corrected peak area were 15 to 35 and 21 to 47, respectively. The linearity R² in terms of corrected peak area were >0.999. Interday precisions (%RSD, n = 6) were 3.3–4.0% for corrected peak area and 2.0–3.0% for migration time. Application to spiked real sample is also presented.     

A two-stage solution approach for the Directed Rural Postman Problem with Turn Penalties

In this paper, we consider the Directed Rural Postman Problem with Turn Penalties (DRPP-TP). A solution is a tour that traverses all required arcs of the graph. The total cost of the tour is the sum of the lengths of the traversed arcs plus the penalties associated with the turns. One solution approach involves transforming the arc routing problem into an equivalent node routing problem. An alternative direct approach (without graph transformation) that involves two stages has been proposed in the literature. In the first part of this paper, we investigate the applicability of the direct approach. We identify several characteristics of the input instance that make this approach effective and present several limitations of this approach. In the second part of this paper, we describe an integer linear program that is combined with a local search algorithm. This combination produces high-quality solutions to the DRPP-TP in a reasonable amount of computing time.     

Heavy traffic analysis of a storage model with long range dependent On/Off sources

We consider a fluid queueing system with infinite storage capacity and constant output rate offered a superposition ofN identical On/Off sources, where the ratio of input to output rate is small. The On and/or Off periods have heavy tailed distributions with infinite variance, giving rise to Long Range Dependence in the arrival process. In the limit of a large number of sources and high load, it is shown that the tail of the stationary queue content distribution is Weibullian, implying much larger queue contents than in the classical case of exponential tails. Noting that similar results were recently found by I. Norros for a storage system input by a Fractional Brownian Motion, we then show how the two models are related, thus providing a further physical motivation for the Fractional Brownian Motion model.     

One‐Electron Oxidation and Sensitivity of Uric Acid in On‐Line Electrochemistry and in Electrospray Ionization Mass Spectrometry

The sensitivity of detection of uric acid (H₂U) in positive ion mode electrospray ionization mass spectrometry (ESI MS) was enhanced by uric acid oxidation during electrospray ionization. With a carrier solution of pH 6.3>pK_a1=5.4 of H₂U, protonated unoxidized uric acid [H₂U+H]⁺ (m/z 169) was detected together with the protonated uric acid dimer [2H₂U+H]⁺ (m/z 337). The dimer likely forms by 1e^? oxidation of urate (HU^?) followed by rapid radical dimerization. A covalent structure of the dimer was verified by H/D exchange experiments. Efficiency of 2e^?, 2H⁺ oxidation of uric acid is low during ESI in pH 6.3 carrier solution and improves when a low on‐line electrochemical cell voltage is floated on the high voltage of the ES in on‐line electrochemistry ESI MS (EC/ESI MS). The intensity of the uric acid dimer decreases with an increase in the low applied voltage. In a carrier solution with 0.1 M KOH, pH 12.7>pK_a2=9.8 of H₂U, allantoin (Allnt) (MW 158.04), the final 2e^?, 2H⁺ oxidation product of uric acid, was detected as a potassium complex [K(Allnt)+K]⁺ (m/z 235) and the [2H₂U+H]⁺ dimer was not detected. In direct ESI MS analysis of 1000‐fold diluted urine [NaHU+H]⁺ (pK_sp NaHU=4.6) was detected in 40/60 (vol%) water/methanol, 1 mM NH₄Ac, pH ca. 6.3 carrier solution. A new configuration of the ESI MS instrument with a cone‐shaped capillary inlet significantly enhanced sensitivity in ESI and EC/ESI MS measurements of uric acid.     

Ultrasensitive analysis of glucose in serum by capillary electrophoresis with LIF detection in combination with signal amplification strategies and on‐column enzymatic assay

A highly specific and sensitive method for glucose quantification in human serum samples based on on‐column enzymatic assay is described. In this method, the head of the capillary was used as a nanoliter‐microreactor, the diluted samples spiked with a novel fluorogenic reagent named 2‐[6‐(4′‐amino) phenoxy‐3H‐xanthen‐3‐on‐9‐yl] benzoic acid (APF), and the mixed enzyme solutions of glucose oxidase (GOx) and horseradish peroxidase (HRP), were individually injected into the capillary. Hydrogen peroxide (H₂O₂) generated in situ by catalytic reaction between GOx and glucose, activates APF in the presence of HRP to form a highly fluorescent product, which was electrophoretically separated from the unreacted APF and detected by the LIF detector. The proposed method allowed the determination of glucose down to 10 nM in real samples, with RSD values lower than 3.5%, which also has the potential for measurements of multicomponents in many other systems including measurement of α‐glucosidase activity and screening for its inhibitors.     

Optimization of a nano‐enzymatic reactor for on‐line tryptic digestion of polypeptide conjugates by capillary electrophoresis

This work aims at studying the optimization of an on‐line capillary electrophoresis (CE)‐based tryptic digestion methodology for the analysis of therapeutic polypeptides (PP). With this methodology, a mixture of surrogate peptide fragments and amino acid were produced on‐line by trypsin cleavage (enzymatic digestion) and subsequently analyzed using the same capillary. The resulting automation of all steps such as injection, mixing, incubation, separation and detection minimizes the possible errors and saves experimental time. In this paper, we first study the differents parameters influencing PP cleavage inside the capillary (plug length, reactant concentration, incubation time, diffusion and electrophoretic plugs mixing). In a second part, the optimization of the electrophoretic separation conditions of generated hydrolysis products (nature, pH and ionic strength (I) of the background electrolyte (BGE)) is described. Using the optimized conditions, excellent repeatability was obtained in terms of separation (migration times) and proteolysis (number of products from enzymatic hydrolysis and corresponding amounts) demonstrating the robustness of the proposed methodology.