Analysis of barbiturates in human serum and urine by high-performance capillary electrophoresis-micellar electrokinetic capillary chromatography with on-column multi-wavelength detection

The analysis of barbiturates in human serum (or plasma) and urine by high-performance capillary electrophoresis-electrokinetic capillary chromatography with on-column fast-scanning multi-wavelength detection is discussed. The use of a buffer of ca. pH 8 and containing sodium dodecyl sulphate provides a medium suitable for fast and high-resolution separations of barbiturates. Seven barbiturates are characterized by their retention and absorption spectra between 195 and 320 nm. Comparison of these computer-stored data with those of unknown samples is shown to allow the identification of barbiturates in samples of patients undergoing pharmacotherapy and in toxicological urine and serum specimens. Three-dimensional electropherograms provide reliable information on the requirement and suitability of sample pretreatment procedures. With urine, extraction of barbiturates prior to analysis is necessary. With human serum several barbiturates, including phenobarbital, are shown to elute in an interference-free window in front of uric acid and the proteins, allowing these substances to be determined by direct sample injection. The need for multi-wavelength detection over a relatively wide wavelength range as a means of peak confirmation in electrokinetic capillary analyses is demonstrated and limitations of this technique for compounds with similar retention behaviour and absorption spectra are discussed.     

Analysis of the disaccharides derived from hyaluronic acid and chondroitin sulfate by capillary electrophoresis with sample stacking

CE conditions for monitoring the unsaturated disaccharides of hyaluronic acid (di-HA) and chondroitin sulfate (di-CS) using an alkaline tetraborate buffer, electrokinetic sample injection, and UV absorption detection at 232 nm are reported. Separations were performed in an uncoated fused-silica capillary having reversed polarity and reversed electroosmosis generated with the addition of CTAB to the buffer. The influence of various separation parameters, including the concentration of CTAB, buffer pH, concentration of tetraborate, and applied voltage, on the resolution of the two disaccharides was investigated. Baseline separation was obtained with 25 mM tetraborate at pH 10.0 and having 0.05 mM CTAB. Chloride and phosphate in the sample are beneficial for the stacking of the disaccharides, with di-HA forming a much sharper peak than di-CS. Using samples prepared in 25 mM Tris-HCl (pH 7.5) and electrokinetic injection at the cathode at -10 kV for 40 s, linear relationships between the corrected peak area and the concentration of the disaccharides have been found in the ranges of 1.0-400.0 and 0.1-1.0 microg/mL (0.2-1.0 microg/mL for di-CS), with correlation coefficients being >0.9933 in all cases. The RSDs of detection times and corrected peak areas were between 1.13-1.24 and 1.57-2.13%, respectively. Applied to human serum samples that were prepared by ethanol precipitation and depolymerization of the two polysaccharides with chondroitinase ABC reveals comigration of endogenous compounds with di-HA and a sample-dependent detection time. The di-HA content in the serum sample can be estimated via subtraction of the blank peak that is obtained without enzymatic hydrolysis.     

Head-column field-amplified sample stacking in presence of siphoning. Application to capillary electrophoresis-electrospray ionization mass spectrometry of opioids in urine

Capillary electrophoresis (CE) with head-column field-amplified sample stacking (FASS) in presence of a water plug inserted at the capillary tip is a robust approach providing a more than 1000-fold sensitivity enhancement when applied to low-conductivity samples that are analyzed in an integrated instrument. Employing modular systems comprising a small hydrodynamic buffer flow (siphoning) towards the capillary end and featuring UV absorption or electrospray ionization mass spectrometric (MS) detection, insertion of a water plug is demonstrated to deteriorate the performance of head-column FASS or making it unfunctional. Electroinjection in the absence of the water plug can be employed instead and is shown to provide a ng/ml sensitivity when applied to low conductivity samples. With some suction of sample into the capillary during electroinjection, contamination of the sample vial with buffer is thereby largely avoided. Electroinjection applied to the CE-ion trap MS-MS and MS-MS-MS analysis of twofold diluted urines, urinary solid-phase extracts and urinary liquid-liquid extracts is shown to provide much improved sensitivity compared to hydrodynamic injection of these samples. With electroinjection from diluted urine and urinary solid-phase extracts, the presence of free opioids and their glucuronic acid conjugates can be unambiguously confirmed in urines that were collected after single-dose administration of small amounts of opioids (tested with about 7 mg codeine and 25 mg dihydrocodeine, respectively). Thus, CE-multiple MS with direct electroinjection of opioids from untreated urines could prove to become a rapid and simple approach for unambiguous urinary testing of drug abuse. Procedures leading to the reduction of siphoning in modular CE setups are briefly discussed as well.     

Determination of gamma-hydroxybutyric acid in human urine by capillary electrophoresis with indirect UV detection and confirmation with electrospray ionization ion-trap mass spectrometry

γ-Hydroxybutyric acid (GHB), a minor metabolite or precursor of γ-aminobutyric acid (GABA), acts as a neurotransmitter/neuromodulator via binding to GABA receptors and to specific presynaptic GHB receptors. Based upon the stimulatory effects, GHB is widely abused. Thus, there is great interest in monitoring GHB in body fluids and tissues. We have developed an assay for urinary GHB that is based upon liquid–liquid extraction and capillary zone electrophoresis (CZE) with indirect UV absorption detection. The background electrolyte is composed of 4 mM nicotinic acid (compound for indirect detection), 3 mM spermine (reversal of electroosmosis) and histidine (added to reach a pH of 6.2). Having a 50 μm I.D. capillary of 40 cm effective length, 1-octanesulfonic acid as internal standard, solute detection at 214 nm and a diluted urine with a conductivity of 2.4 mS/cm, GHB concentrations ≥2 μg/ml can be detected. Limit of detection (LOD) and limit of quantitation (LOQ) were determined to be dependent on urine concentration and varied between 2–24 and 5–60 μg/ml, respectively. Data obtained suggest that LOD and LOQ (both in μg/ml) can be estimated with the relationships 0.83 κ and 2.1 κ, respectively, where κ is the conductivity of the urine in mS/cm. The assay was successfully applied to urines collected after administration of 25 mg sodium GHB/kg body mass. Negative electrospray ionization ion-trap tandem mass spectrometry was used to confirm the presence of GHB in the urinary extract via selected reaction monitoring of the m/z 103.1→m/z 85.1 precursor–product ion transition. Independent of urine concentration, this approach meets the urinary cut-off level of 10 μg/ml that is required for recognition of the presence of exogenous GHB. Furthermore, data obtained with injection of plain or diluted urine indicate that CZE could be used to rapidly recognize GHB amounts (in μg/ml) that are ≥ 4 κ.     

Determination of thiopental in human serum and plasma by high-performance capillary electrophoresis-micellar electrokinetic chromatography.

The quantitation of thiopental in human serum and plasma was investigated using high-performance capillary electrophoresis (HPCE) in a micellar configuration and the results were compared with reversed-phase high-performance liquid chromatography (HPLC). Thiopental and an internal standard (carbamazepine for HPCE and thiamylal for HPLC) were extracted from serum or plasma using pentane and a phosphate buffer (pH 6.4). HPCE analysis took place in a phosphate-borate buffer with 50 mM sodium dodecyl sulphate using an automated instrument and HPLC was performed with a C8 column and a mobile phase of phosphate buffer-acetonitrile (65:35, v/v). HPCE and HPLC data from 66 patient samples compared well based on linear regression analysis. However, estimates obtained with the inclusion of the internal standard were lower than those based on the sample peak only. This example allows the elucidation of the advantages of using HPCE as an assay methodology for the therapeutic monitoring of thiopental and other drugs.     

An Alternative to Automatic Titration

Automatic titrators are designed to do exactly what a lab-technician used to do. Much more sophisticated automation of volumetry is possible. Omegaphoresis [3] in buffer-free [2] sample solutions automatically creates a stationary multiple component titration curve or zoned pattern with normalized concentrations of each separated species. An automatic measurement of each zone length yields their quantity. Simultaneous automatic detection of all the zones in a 10–20 component solution in less than 2 minutes, with a precision of ±2%, a required amount of the order of less than one nanomol and a resolution of ±pK < 0.01 replaces acid-base, complexometric, and certain types of redox-titrimetry. An option of the method allows identification of the components as well.     

Electrokinetic characterization of poly(dimethylsiloxane) microchannels

This paper characterizes the basic electrokinetic phenomena occurring within native poly(dimethylsiloxane) (PDMS) microchannels. Using simple buffers and current measurements, current density and electroosmosis data were determined in trapezoidal, reversibly sealed PDMS/PDMS and hybrid PDMS/glass channels with a cross-sectional area of 1035.5 microm(2) and about 6 cm length. This data was then compared to that obtained in an air-thermostated 50 microm inner diameter (1963.5 microm(2) cross-sectional area) fused-silica (FS) capillary of 70 cm length. Having a pH 7.8 buffer with an ionic strength (I) of 90 mM, Ohms's law was observed in the microchannels with electric field strengths of up to about 420 V/cm, which is about twice as high as for the FS capillary. The electroosmotic mobility (micro(EO)) in PDMS and FS is shown to exhibit the same general dependences on I and pH. For all configurations tested, the experimentally determined micro(EO) values were found to correlate well with the relationship micro(EO) = a + b log(I), where a and b are coefficients that are determined via nonlinear regression analysis. Electroosmotic fluid pumping in native PDMS also follows a pH dependence that can be estimated with a model based upon the ionization of silanol. Compared to FS, however, the magnitude of the electroosmotic flow in native PDMS is 50-70% smaller over the entire pH range and is difficult to maintain at acidic pH values. Thus, the origin of the negative charge at the inner wall of PDMS, glass, and FS appears to be similar but the density is lower for PDMS than for glass and FS.     

Recycling and screen-segmented column isotachophoresis, two free-fluid approaches for fractionation of proteins.

Recycling and screen-segmented column isotachophoresis (ITP), two approaches for the milligrams to grams preparative-scale purification of proteins, are discussed and compared. Recycling ITP was performed in a recycling free-flow focusing apparatus. In this process, fluid flows rapidly through a narrow channel and the effluent from each channel is reinjected into the electrophoresis chamber through the corresponding input port. The residence time in the cell is of the order of 1 s per single pass, which does not allow complete separation, so recycling is essential to attain the steady state. Immobilization of the advancing zone structure is obtained via a controlled counterflow. Thirty fractions of about 4 ml each are obtained. Column ITP was executed in a Rotofor apparatus and in a similar column operated vertically and without rotation. These instruments feature a screen-segmented annular separation space with twenty subcompartments of about 2 ml each. With both approaches, the collected fractions were analysed separately for conductivity, pH and UV absorbance. Selected fractions were characterized by analytical electrophoretic methods. Examples presented include the cationic and anionic ITP behaviour of model proteins, including bovine serum albumin, ovalbumin and ribonuclease A, and the ITP removal of the major impurities from a commercial ovalbumin sample. These examples revealed that the screen-segmented column is suitable for ITP protein purification and operates optimally in a horizontal rotating mode and without internal cooling. The recycling experiments showed that counterflow improves separation and the steady-state patterns are dependent on the fluid layer thickness in the separation cell but, with a given gap, essentially independent of applied current and recycling pump rate.     

Determination of hexacyanoferrate based on its catalytic influence on the oxidation of p-phenylenediamines by iron(III)

Summary Both hexacyanoferrate(III) and hexacyanoferrate(II) catalyze the oxidation of p-phenylenediamines by iron(III)_aq. The rate of this reaction in the presence of a sample with an unknown amount of hexacyanoferrate is compared with the reaction rate of solutions containing well defined concentrations of this substance. In this way, hexacyanoferrate can be determined photometrically down to <10^–9 mol/l. Although this procedure is very sensitive, the analysis can be performed with a simple photometer. Absorbance changes >0.2 can easily be obtained in 1 cm cuvettes, even at extremely small concentrations of hexacyanoferrate, because it is not an absorbance proportional to the concentration of hexacyanoferrate but rather the formation rate of p-semiquinonediimine which enables the quantitative determination of hexacyanoferrate.     

Isotachophoretic zone formation of serum albumin in different free fluid electrophoresis instruments

The isotachophoretic behavior of a model protein, serum albumin, was examined (i) by computer simulation, (ii) by capillary isotachophoresis in HPE 100 and Tachophor 2127, (iii) by continuous flow isotachophoresis in Elphor VaP 22 and the BIO-STREAM Separator and (iv) by recycling isotachophoresis in an apparatus of our own design. Variations in monitored zone shapes can be explained by differences in engineering aspects and fluid stabilization principles of the instruments.